Myostatin binding agents, nucleic acids encoding the same, and methods of treatment

ABSTRACT

The present invention provides binding agents comprising peptides capable of binding myostatin and inhibiting its activity. In one embodiment the binding agent comprises at least one myostatin-binding peptide attached directly or indirectly to at least one vehicle such as a polymer or an Fc domain. The binding agents of the present invention produced increased lean muscle mass when administered to animals and decreased fat to muscle ratios. Therapeutic compositions containing the binding agents of the present invention are useful for treating muscle-wasting disorders and metabolic disorders including diabetes and obesity.

This application is a continuation of U.S. patent application Ser. No.13/310,661, filed Dec. 2, 2011 (abandoned), which is a divisional ofU.S. patent application Ser. No. 12/806,880, filed Aug. 23, 2010, nowU.S. Pat. No. 8,071,538, which is a divisional of U.S. patentapplication Ser. No. 12/322,369, filed Jan. 30, 2009, now U.S. Pat. No.7,803,923, which is a divisional of U.S. patent application Ser. No.10/742,379, filed Dec. 19, 2003, now U.S. Pat. No. 7,511,012, whichhereby claims benefit of U.S. provisional application Ser. No.60/435,923, filed Dec. 20, 2002, the entire disclosure of each of theabove applications is relied upon and incorporated by reference herein.

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-828-US-DIV3.txt created Oct. 20, 2011, which is 190 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

Throughout this application various publications are referenced withinparentheses or brackets. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

FIELD OF THE INVENTION

The invention relates to growth factors and in particular to the growthfactor myostatin and agents which bind myostatin and inhibit itsactivity.

BACKGROUND

Myostatin, also known as growth/differentiation factor 8 (GDF-8), is atransforming growth factor-β (TGF-β) family member known to be involvedin regulation of skeletal muscle mass. Most members of the TGF-β-GDFfamily are expressed non-specifically in many tissue types and exert avariety of pleitrophic actions. However, myostatin is largely expressedin the cells of developing and adult skeletal muscle tissue and plays anessential role in negatively controlling skeletal muscle growth(McPherron et al. Nature (London) 387, 83-90 (1997)). Recent studies,however, indicate that low levels of myostatin expression can bemeasured in cardiac, adipose and pre-adipose tissues.

The myostatin protein has been highly conserved evolutionarily(McPherron et al. PNAS USA 94:12457-12461 (1997)). The biologicallyactive C-terminal region of myostatin has 100 percent sequence identitybetween human, mouse, rat, cow, chicken, and turkey sequences. Thefunction of myostatin also appears to be conserved across species aswell. This is evident from the phenotypes of animals having a mutationin the myostatin gene. Two breeds of cattle, the Belgian Blue (HansetR., Muscle Hypertrophy of Genetic Origin and its Use to Improve BeefProduction, eds, King, J. W. G. & Menissier, F. (Nijhoff, The Hague, TheNetherlands) pp. 437-449) and the Piedmontese (Masoero, G. & Poujardieu,B, Muscle Hypertrophy of Genetic Origin and its Use to Improve BeefProduction., eds, King, J. W. G. & Menissier, F. (Nijhoff, The Hague,The Netherlands) pp. 450-459) are characterized by a “double muscling”phenotype and increase in muscle mass. These breeds were shown tocontain mutations in the coding region of the myostatin gene (McPherronet al. (1997) supra). In addition, mice containing a targeted deletionof the gene encoding myostatin (Mstn) demonstrate a dramatic increase inmuscle mass without a corresponding increase in fat. Individual musclesof Mstn^(−/−) mice weigh approximately 100 to 200 percent more thanthose of control animals as a result of muscle fiber hypertrophy andhyperplasia (Zimmers et al. Science 296, 1486 (2002)).

Administration of myostatin to certain strains of mice has been shown tocreate a condition similar to muscle wasting disorders found associatedwith cancer, AIDS, and muscular dystrophy, for example. Myostatinadministered as myostatin-producing CHO cells to athymic nude miceresulted in a wasting effect with a high degree of weight loss, adecrease of as much as 50% of skeletal muscle mass in addition to fatwasting, and severe hypoglycemia (Zimmers et al. supra).

Loss of myostatin appears to result in the retention of muscle mass andreduction in fat accumulation with aging. It has been shown thatage-related increases in adipose tissue mass and decrease in muscle masswere proportional to myostatin levels, as determined by a comparison offat and muscle mass in Mstn^(−/−) when compared with Mstn^(−/−) adultknockout mice (McFerron et al. J. Clin. Invest 109, 595 (2002)).Mstn^(−/−) mice showed decreased fat accumulation with age compared withMstn^(−/−) mice.

In addition myostatin may play a role in maintaining blood glucoselevels and may influence the development of diabetes in certain cases.It is known that, for example, skeletal muscle resistance toinsulin-stimulated glucose uptake is the earliest known manifestation ofnon-insulin-dependent (type 2) diabetes mellitus (Corregan et al.Endocrinology 128:1682 (1991)). It has now been shown that the lack ofmyostatin partially attenuates the obese and diabetes phenotypes of twomouse models, the agouti lethal yellow (A^(y)) (Yen et al. FASEB J.8:479 (1994)), and obese (Lep^(ob/ob)). Fat accumulation and total bodyweight of the AY^(y/a), Mstn^(−/−) double mutant mouse was dramaticallyreduced compared with the A^(y/a) Mstn^(−/−) mouse (McFerron et al.,(2002) supra). In addition, blood glucose levels in the A^(y/a),Mstn^(−/−) mice was dramatically lower than in A^(y/a) Mstn^(+/+) micefollowing exogenous glucose load, indicating that the lack of myostatinimproved glucose metabolism. Similarly Lep^(ob/ob) Mstn^(−/−) miceshowed decreased fat accumulation when compared with the Lep^(ob/ob)Mstn^(1/1) phenotype.

Therefore, there is considerable evidence from the phenotypes ofover-expressing and knockout animals that myostatin may play a role incontributing to a number of metabolic disorders including disordersresulting in muscle wasting, diabetes, obesity and hyperglycemia.

SUMMARY OF THE INVENTION

The present invention is directed to binding agents which bind myostatinand inhibit its activity. The binding agents comprise at least onepeptide capable of binding myostatin. The myostatin-binding peptides arepreferably between about 5 and about 50 amino acids in length, morepreferably between about 10 and 30 amino acids in length, and mostpreferably between about 10 and 25 amino acids in length. In oneembodiment the myostatin-binding peptide comprises the amino acidsequence WMCPP (SEQ ID NO: 633). In another embodiment the myostatinbinding peptides comprise the amino acid sequence Ca₁a₂ Wa₃ WMCPP (SEQID NO: 352), wherein a₁, a₂ and a₃ are selected from a neutralhydrophobic, neutral polar, or basic amino acid. In another embodimentthe myostatin binding peptide comprises the sequence Cb₁b₂ Wb₃ WMCPP(SEQ ID NO: 353), wherein b₁ is selected from any one of the amino acidsT, I, or R; b₂ is selected from any one of R, S, Q; b₃ is selected fromany one of P, R and Q, and wherein the peptide is between 10 and 50amino acids in length, and physiologically acceptable salts thereof. Inanother embodiment, the myostatin binding peptide comprises the formula:c₁c₂c₃c₄c₅c₆ Cc₇c₈ Wc₉ WMCPPc₁₀c₁₁c₁₂C₁₃ (SEQ ID NO: 354),

wherein:

c₁ is absent or any amino acid;

c₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₄ is absent or any amino acid;

c₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

c₇ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₈ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₉ is a neutral hydrophobic, neutral polar or basic amino acid; and

c₁₀ to c₁₃ is any amino acid; and wherein the peptide is between 20 and50 amino acids in length, and physiologically acceptable salts thereof.

A related embodiment the myostatin binding peptide comprises theformula:d₁d₂d₃d₄d₅d₆ Cd₇d₈ Wd₉ WMCPPd₁₀d₁₁d₁₂d₁₃ (SEQ ID NO: 355),

wherein

d₁ is absent or any amino acid;

d₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₄ is absent or any amino acid;

d₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

d₇ is selected from any one of the amino acids T, I, or R;

d₈ is selected from any one of R, S, Q;

d₉ is selected from any one of P, R and Q, and

d₁₀ to d₁₃ is selected from any amino acid,

and wherein the peptide is between 20 and 50 amino acids in length, andphysiologically acceptable salts thereof.

Additional embodiments of binding agents comprise at least one of thefollowing peptides:

(1) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence WYe₁e₂ Ye₃ G, (SEQ ID NO: 356),

wherein e₁ is P, S or Y,

e₂ is C or Q, and

e₃ is G or H, wherein the peptide is between 7 and 50 amino acids inlength, and physiologically acceptable salts thereof;

(2) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence f₁ EMLf ₂ SLf₃f₄ LL, (SEQ ID NO: 455),

wherein f₁ is M or I,

f₂ is any amino acid,

f₃ is L or F,

f₄ is E, Q or D;

and wherein the peptide is between 7 and 50 amino acids in length, andphysiologically acceptable salts thereof;

(3) a peptide capable of binding myostatin wherein the peptide comprisesthe sequence Lg₁g₂ LLg₃g₄ L, (SEQ ID NO: 456), wherein

g₁ is Q, D or E,

g₂ is 5, Q, D or E,

g₃ is any amino acid,

g₄ is L, W, F, or Y, and wherein the peptide is between 8 and 50 aminoacids in length, and physiologically acceptable salts thereof;

(4) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence h₁h₂h₃h₄h₅h₆h₇h₈h₉ (SEQ ID NO: 457), wherein

h₁ is R or D,

h₂ is any amino acid,

h₃ is A, T S or Q,

h₄ is L or M,

h₅ is L or S,

h₆ is any amino acid,

h₇ is F or E,

h₈ is W, F or C,

h₉ is L, F, M or K, and wherein the peptide is between 9 and 50 aminoacids in length, and physiologically acceptable salts thereof.

In one embodiment, the binding agents of the present invention furthercomprise at least one vehicle such as a polymer or an Fc domain, and mayfurther comprise at least one linker sequence. In this embodiment, thebinding agents of the present invention are constructed so that at leastone myostatin-binding peptide is attached to at least one vehicle. Thepeptide or peptides are attached directly or indirectly through a linkersequence, to the vehicle at the N-terminal, C-terminal or an amino acidsidechain of the peptide. In this embodiment, the binding agents of thepresent invention have the following generalized structure:

(X¹)_(a)—F¹—(X²)_(b),

or multimers thereof;

wherein F¹ is a vehicle; and X¹ and X² are each independently selectedfrom

-(L¹)_(c)-P¹;

-(L¹)_(c)-P¹-(L²)_(d)-P²;

-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³;

and -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;

wherein P¹, P², P³, and P⁴ are peptides capable of binding myostatin;and

L¹, L², L³, and L⁴ are each linkers; and a, b, c, d, e, and f are eachindependently 0 or 1,

provided that at least one of a and b is 1, and physiologicallyacceptable salts thereof.

In various embodiments of binding agents having this generalizedstructure, the peptides P¹, P², P³, and P⁴ can be independently selectedfrom one or more of any of the peptides comprising the sequencesprovided above. P¹, P², P³, and P⁴ are independently selected from oneor more peptides comprising any of the following sequences: SEQ ID NO:633, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQID NO: 356, SEQ ID NO: 455, SEQ ID NO: 456, or SEQ ID NO: 457.

In a further embodiment, the binding agents comprise peptides fused toan Fc domain, either directly or indirectly, thereby providingpeptibodies. The peptibodies of the present invention display a highbinding affinity for myostatin and can inhibit the activity of myostatinas demonstrated both in vitro using cell based assays and in animals.

The present invention also provides nucleic acid molecules comprisingpolynucleotides encoding the peptides, peptibodies, and peptide andpeptibody variants and derivatives of the present invention.

The present invention provides pharmaceutically acceptable compositionscomprising one or more binding agents of the present invention.

The binding agents of the present invention inhibit myostatin activityin vitro and in vivo. The binding agents of the present inventionincrease lean muscle mass in a treated animal and decreases fat mass asa percentage of body weight of the animal. The myostatin binding agentsof the present invention increase muscular strength in treated animalmodels.

The present invention provides methods of inhibiting myostatin activityin animals including humans by administering an effective dosage of oneor more binding agents to the subject. The present invention providesmethods of increasing lean muscle mass in animals including humans byadministering an effective dosage of one or more binding agents. Thepresent invention further provides methods of treating myostatin-relateddisorders by administering a therapeutically effective dosage of one ormore myostatin binding agents in a pharmaceutically acceptablecomposition to a subject. The present invention provides methods oftreating muscle wasting disorders including muscular dystrophy, musclewasting due to cancer, AIDS, rheumatoid arthritis, renal failure,uremia, chronic heart failure, age-related sarcopenia, prolongedbed-rest, spinal chord injury, stroke, bone fracture. The presentinvention also provides methods of treating metabolic disordersincluding obesity, diabetes, hyperglycemia, and bone loss.

The present invention also provides a method of increasing muscle massin food animals by administering an effective dosage of one or moremyostatin binding agents to the animal.

The present invention provides assays utilizing one or more myostatinbinding agents to identify and quantitate myostatin in a sample. Theassays may be diagnostic assays for measuring or monitoring myostatinlevels in individuals with a myostatin related disorder or disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows myostatin activity as measured by expressed luciferaseactivity (y-axis) vs. concentration (x-axis) for the TN8-19 peptideQGHCTRWPWMCPPY (Seq ID No: 32) and the TN8-19 peptibody (pb) todetermine the IC₅₀ for each using the C2C12 pMARE luciferase assaydescribed in the Examples below. The peptibody has a lower IC₅₀ valuecompared with the peptide.

FIG. 2 is a graph showing the increase in total body weight for CD1nu/nu mice treated with increasing dosages of the 1× mTN8-19-21peptibody over a fourteen day period compared with mice treated with ahuFc control, as described in Example 8.

FIG. 3A shows the increase in the mass of the gastrocnemius muscle massat necropsy of the mice treated in FIG. 2 (Example 8). FIG. 3B shows theincrease in lean mass as determined by NMR on day 0 compared with day 13of the experiment described in Example 8.

FIG. 4 shows the increase in lean body mass as for CD1 nu/nu micetreated with biweekly injections of increasing dosages of 1× mTN8-19-32peptibody as determined by NMR on day 0 and day 13 of the experimentdescribed in Example 8.

FIG. 5A shows the increase in body weight for CD1 nu/nu mice treatedwith biweekly injections of 1× mTN8-19-7 compared with 2× mTN8-19-7 andthe control animal for 35 days as described in Example 8. FIG. 5B showsthe increase in lean carcass weight at necropsy for the 1× and 2×versions at 1 mg/kg and 3 mg/kg compared with the animals receiving thevehicle (huFc) (controls).

FIG. 6A shows the increase in lean muscle mass vs. body weight for agedmdx mice treated with either affinity matured 1× mTN8-19-33 peptibody orhuFc vehicle at 10 mg/kg subcutaneously every other day for threemonths. FIG. 6B shows the change in fat mass compared to body weight asdetermined by NMR for the same mice after 3 months of treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides binding agents capable of bindingmyostatin and inhibiting its activity. The myostatin binding agents canbe used in assays, to identify, quantitate, or monitor the level ofmyostatin in an animal. The myostatin binding agents of the presentinvention reduce myostatin activity. The myostatin binding agents of thepresent invention increase lean muscle mass in animals, decrease fatmass as a percentage of body weight, and increase muscle strength. Themyostatin binding agents of the present invention can be used to treat avariety of metabolic disorders in which myostatin plays a role,including muscle wasting disorders such as muscular dystrophies, musclewasting due to cancer, AIDS, rheumatoid arthritis, renal failure,uremia, chronic heart failure, prolonged bed-rest, spinal chord injury,stroke, and age-related sarcopenia as well as other metabolic disordersincluding diabetes, obesity, hyperglycemia, and bone loss, byadministering a therapeutic dosage of one or more binding agents in apharmaceutically acceptable composition to a subject.

Myostatin

Myostatin, a growth factor also known as GDF-8, is known to be anegative regulator of skeletal muscle tissue. Myostatin is synthesizedas an inactive preproprotein which is activated by proteolyic cleavage(Zimmers et al., supra (2002)). The precurser protein is cleaved toproduce an NH₂-terminal inactive prodomain and an approximately 109amino acid COOH-terminal protein in the form of a homodimer of about 25kDa, which is the mature, active form (Zimmers et al, supra (2002)). Itis now believed that the mature dimer circulates in the blood as aninactive latent complex bound to the propeptide (Zimmers et al, supra(2002).

As used herein the term “full-length myostatin” refers to thefull-length human preproprotein sequence described in McPherson et al.supra (1997), as well as related full-length polypeptides includingallelic variants and interspecies homologs which are also described inMcPherron et al. (1997). As used herein, the term “prodomain” or“propeptide” refers to the inactive NH₂-terminal protein which iscleaved off to release the active COOH-terminal protein. As used hereinthe term “myostatin” or “mature myostatin” refers to the mature,biologically active COOH-terminal polypeptide, in monomer, dimer,multimeric form or other form. “Myostatin” or “mature myostatin” alsorefers to fragments of the biologically active mature myostatin, as wellas related polypeptides including allelic variants, splice variants, andfusion peptides and polypeptides. The mature myostatin COOH-terminalprotein has been reported to have 100% sequence identity among manyspecies including human, mouse, chicken, porcine, turkey, and rat (Leeet al., PNAS 98, 9306 (2001)). Myostatin may or may not includeadditional terminal residues such as targeting sequences, or methionineand lysine residues and/or tag or fusion protein sequences, depending onhow it is prepared.

As used herein the term “capable of binding to myostatin” or “having abinding affinity for myostatin” refers to a binding agent or peptidewhich binds to myostatin as demonstrated by as the phage ELISA assay,the BIAcore® or KinExA™ assays described in the Examples below.

As used herein, the term “capable of modifying myostatin activity”refers to the action of an agent as either an agonist or an antagonistwith respect to at least one biological activity of myostatin. As usedherein, “agonist” or “mimetic” activity refers an agent havingbiological activity comparable to a protein that interacts with theprotein of interest, as described, for example, in Internationalapplication WO 01/83525, filed May 2, 2001, which is incorporated hereinby reference.

As used herein, the term “inhibiting myostatin activity” or “havingantagonist activity” refers to the ability of a peptide or binding agentto reduce or block myostatin activity or signaling as demonstrated or invitro assays such as, for example, the pMARE C2C12 cell-based myostatinactivity assay or by In vivo animal testing as described below.

Structure of Myostatin Binding Agents

In one embodiment, the binding agents of the present invention compriseat least one myostatin binding peptide covalently attached to at leastone vehicle such as a polymer or an Fc domain. The attachment of themyostatin-binding peptides to at least one vehicle is intended toincrease the effectiveness of the binding agent as a therapeutic byincreasing the biological activity of the agent and/or decreasingdegradation in vivo, increasing half-life in vivo, reducing toxicity orimmunogenicity in vivo. The binding agents of the present invention mayfurther comprise a linker sequence connecting the peptide and thevehicle. The peptide or peptides are attached directly or indirectlythrough a linker sequence to the vehicle at the N-terminal, C-terminalor an amino acid sidechain of the peptide. In this embodiment, thebinding agents of the present invention have the following structure:(X¹)_(a)—F¹—(X²)_(b),

or multimers thereof;

wherein F¹ is a vehicle; and X¹ and X² are each independently selectedfrom

-(L¹)_(c)-P¹;

-(L¹)_(c)-P¹-(L²)_(d)-P²;

-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³;

and -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;

wherein P¹, P², P³, and P⁴ are peptides capable of binding myostatin;and

L¹, L², L³, and L⁴ are each linkers; and a, b, c, d, e, and f are eachindependently 0 or 1,

provided that at least one of a and b is 1.

Any peptide containing a cysteinyl residue may be cross-linked withanother Cys-containing peptide, either or both of which may be linked toa vehicle. Any peptide having more than one Cys residue may form anintrapeptide disulfide bond, as well.

In one embodiment, the vehicle is an Fc domain, defined below. Thisembodiment is referred to as a “peptibody”. As used herein, the term“peptibody” refers to a molecule comprising an antibody Fc domainattached to at least one peptide. The production of peptibodies isgenerally described in PCT publication WO 00/24782, published May 4,2000, which is herein incorporated by reference. Exemplary peptibodiesare provided as 1× and 2× configurations with one copy and two copies ofthe peptide (attached in tandem) respectively, as described in theExamples below.

Peptides

As used herein the term “peptide” refers to molecules of about 5 toabout 90 amino acids linked by peptide bonds. The peptides of thepresent invention are preferably between about 5 to about 50 amino acidsin length, more preferably between about 10 and 30 amino acids inlength, and most preferably between about 10 and 25 amino acids inlength, and are capable of binding to the myostatin protein.

The peptides of the present invention may comprise part of a sequence ofnaturally occurring proteins, may be randomized sequences derived fromnaturally occurring proteins, or may be entirely randomized sequences.The peptides of the present invention may be generated by any methodsknown in the art including chemical synthesis, digestion of proteins, orrecombinant technology. Phage display and RNA-peptide screening, andother affinity screening techniques are particularly useful forgenerating peptides capable of binding myostatin.

Phage display technology is described, for example, in Scott et al.Science 249: 386 (1990); Devlin et al., Science 249: 404 (1990); U.S.Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731,issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996;U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665,issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16,1998, each of which is incorporated herein by reference. Using phagelibraries, random peptide sequences are displayed by fusion with coatproteins of filamentous phage. Typically, the displayed peptides areaffinity-eluted either specifically or non-specifically against thetarget molecule. The retained phages may be enriched by successiverounds of affinity purification and repropagation. The best bindingpeptides are selected for further analysis, for example, by using phageELISA, described below, and then sequenced. Optionally, mutagenesislibraries may be created and screened to further optimize the sequenceof the best binders (Lowman, Ann Rev Biophys Biomol Struct 26:401-24(1997)).

Other methods of generating the myostatin binding peptides includeadditional affinity selection techniques known in the art. A peptidelibrary can be fused in the carboxyl terminus of the lac repressor andexpressed in E. coli. Another E. coli-based method allows display on thecell's outer membrane by fusion with a peptidoglycan-associatedlipoprotein (PAL). Hereinafter, these and related methods arecollectively referred to as “E. coli display.” In another method,translation of random RNA is halted prior to ribosome release, resultingin a library of polypeptides with their associated RNA still attached.Hereinafter, this and related methods are collectively referred to as“ribosome display.” Other methods employ chemical linkage of peptides toRNA. See, for example, Roberts and Szostak, Proc Natl Acad Sci USA, 94:12297-303 (1997). Hereinafter, this and related methods are collectivelyreferred to as “RNA-peptide screening.” Yeast two-hybrid screeningmethods also may be used to identify peptides of the invention that bindto myostatin. In addition, chemically derived peptide libraries havebeen developed in which peptides are immobilized on stable,non-biological materials, such as polyethylene rods or solvent-permeableresins. Another chemically derived peptide library uses photolithographyto scan peptides immobilized on glass slides. Hereinafter, these andrelated methods are collectively referred to as “chemical-peptidescreening.” Chemical-peptide screening may be advantageous in that itallows use of D-amino acids and other analogues, as well as non-peptideelements. Both biological and chemical methods are reviewed in Wells andLowman, Curr Opin Biotechnol 3: 355-62 (1992).

Additionally, selected peptides capable of binding myostatin can befurther improved through the use of “rational design”. In this approach,stepwise changes are made to a peptide sequence and the effect of thesubstitution on the binding affinity or specificity of the peptide orsome other property of the peptide is observed in an appropriate assay.One example of this technique is substituting a single residue at a timewith alanine, referred to as an “alanine walk” or an “alanine scan”.When two residues are replaced, it is referred to as a “double alaninewalk”. The resultant peptide containing amino acid substitutions aretested for enhanced activity or some additional advantageous property.

In addition, analysis of the structure of a protein-protein interactionmay also be used to suggest peptides that mimic the interaction of alarger protein. In such an analysis, the crystal structure of a proteinmay suggest the identity and relative orientation of critical residuesof the protein, from which a peptide may be designed. See, for example,Takasaki et al., Nature Biotech 15:1266 (1977). These methods may alsobe used to investigate the interaction between a targeted protein andpeptides selected by phage display or other affinity selectionprocesses, thereby suggesting further modifications of peptides toincrease binding affinity and the ability of the peptide to inhibit theactivity of the protein.

In one embodiment, the peptides of the present invention are generatedas families of related peptides. Exemplary peptides are represented bySEQ ID NO: 1 through 132. These exemplary peptides were derived througha selection process in which the best binders generated by phage displaytechnology were further analyzed by phage ELISA to obtain candidatepeptides by an affinity selection technique such as phage displaytechnology as described herein. However, the peptides of the presentinvention may be produced by any number of known methods includingchemical synthesis as described below.

The peptides of the present invention can be further improved by theprocess of “affinity maturation”. This procedure is directed toincreasing the affinity or the activity of the peptides and peptibodiesof the present invention using phage display or other selectiontechnologies. Based on a consensus sequence, directed secondary phagedisplay libraries, for example, can be generated in which the “core”amino acids (determined from the consensus sequence) are held constantor are biased in frequency of occurrence. Alternatively, an individualpeptide sequence can be used to generate a biased, directed phagedisplay library. Panning of such libraries under more stringentconditions can yield peptides with enhanced binding to myostatin,selective binding to myostatin, or with some additional desiredproperty. However, peptides having the affinity matured sequences maythen be produced by any number of known methods including chemicalsynthesis or recombinantly. These peptides are used to generate bindingagents such as peptibodies of various configurations which exhibitgreater inhibitory activity in cell-based assays and in vivo assays.

Example 6 below describes affinity maturation of the “first round”peptides described above to produce affinity matured peptides. Exemplaryaffinity matured peptibodies are presented in Tables IV and V. Theresultant 1× and 2× peptibodies made from these peptides were thenfurther characterized for binding affinity, ability to neutralizemyostatin activity, specificity to myostatin as opposed to other TNFβfamily members, and for additional in vitro and in vivo activity, asdescribed below. Affinity-matured peptides and peptibodies are referredto by the prefix “m” before their family name to distinguish them fromfirst round peptides of the same family.

Exemplary first round peptides chosen for further affinity maturationaccording to the present invention included the following peptides:TN8-19 QGHCTRWPWMCPPY (SEQ ID NO: 33), and the linear peptides Linear-2MEMLDSLFELLKDMVPISKA (SEQ ID NO: 104), Linear-15 HHGWNYLRKGSAPQWFEAWV(SEQ ID NO: 117), Linear-17, RATLLKDFWQLVEGYGDN (SEQ ID NO: 119),Linear-20 YREMSMLEGLLDVLERLQHY (SEQ ID NO: 122), Linear-21HNSSQMLLSELIMLVGSMMQ (SEQ ID NO: 123), Linear-24 EFFHWLHNHRSEVNHWLDMN(SEQ ID NO: 126). The affinity matured families of each of these ispresented below in Tables IV and V.

The peptides of the present invention also encompass variants andderivatives of the selected peptides which are capable of bindingmyostatin. As used herein the term “variant” refers to peptides havingone or more amino acids inserted, deleted, or substituted into theoriginal amino acid sequence, and which are still capable of binding tomyostatin. Insertional and substitutional variants may contain naturalamino acids as well as non-naturally occurring amino acids. As usedherein the term “variant” includes fragments of the peptides which stillretain the ability to bind to myostatin. As used herein, the term“derivative” refers to peptides which have been modified chemically insome manner distinct from insertion, deletion, and substitutionvariants. Variants and derivatives of the peptides and peptibodies ofthe present invention are described more fully below.

Vehicles

As used herein the term “vehicle” refers to a molecule that may beattached to one or more peptides of the present invention. Preferably,vehicles confer at least one desired property on the binding agents ofthe present invention. Peptides alone are likely to be removed in vivoeither by renal filtration, by cellular clearance mechanisms in thereticuloendothelial system, or by proteolytic degradation. Attachment toa vehicle improves the therapeutic value of a binding agent by reducingdegradation of the binding agent and/or increasing half-life, reducingtoxicity, reducing immunogenicity, and/or increasing the biologicalactivity of the binding agent.

Exemplary vehicles include Fc domains; linear polymers such aspolyethylene glycol (PEG), polylysine, dextran; a branched chain polymer(see for example U.S. Pat. No. 4,289,872 to Denkenwalter et al., issuedSep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; acholesterol group (such as a steroid); a carbohydrate oroligosaccharide; or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor.

In one embodiment, the myostatin binding agents of the present inventionhave at least one peptide attached to at least one vehicle (F¹, F²)through the N-terminus, C-terminus or a side chain of one of the aminoacid residues of the peptide(s). Multiple vehicles may also be used;such as an Fc domain at each terminus or an Fc domain at a terminus anda PEG group at the other terminus or a side chain.

An Fc domain is one preferred vehicle. As used herein, the term “Fcdomain” encompasses native Fc and Fc variant molecules and sequences asdefined below. As used herein the term “native Fe” refers to anon-antigen binding fragment of an antibody or the amino acid sequenceof that fragment which is produced by recombinant DNA techniques or byenzymatic or chemical cleavage of intact antibodies. A preferred Fc is afully human Fc and may originate from any of the immunoglobulins, suchas IgG1 and IgG2. However, Fc molecules that are partially human, ororiginate from non-human species are also included herein. Native Fcmolecules are made up of monomeric polypeptides that may be linked intodimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucl Acids Res 10: 4071-9). The term “native Fc” as used hereinis used to refer to the monomeric, dimeric, and multimeric forms.

As used herein, the term “Fc variant” refers to a modified form of anative Fc sequence provided that binding to the salvage receptor ismaintained, as described, for example, in WO 97/34631 and WO 96/32478,both of which are incorporated herein by reference. Fc variants may beconstructed for example, by substituting or deleting residues, insertingresidues or truncating portions containing the site. The inserted orsubstituted residues may also be altered amino acids, such aspeptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removalmay avoid reaction with other cysteine-containing proteins present inthe host cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus may betruncated or cysteine residues may be deleted or substituted with otheramino acids (e.g., alanyl, seryl). Even when cysteine residues areremoved, the single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selectedhost cell. For example, one may remove the PA sequence near theN-terminus of a typical native Fc, which may be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One may alsoadd an N-terminal methionyl residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli.

3. A portion of the N-terminus of a native Fc is removed to preventN-terminal heterogeneity when expressed in a selected host cell. Forthis purpose, one may delete any of the first 20 amino acid residues atthe N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) may confer cytolytic response.Such residues may be deleted or substituted with unglycosylated residues(e.g., alanine).

5. Sites involved in interaction with complement, such as the C1qbinding site, are removed. For example, one may delete or substitute theEKK sequence of human IgG1. Complement recruitment may not beadvantageous for the molecules of this invention and so may be avoidedwith such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than asalvage receptor. A native Fc may have sites for interaction withcertain white blood cells that are not required for the fusion moleculesof the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art. See, forexample, Molec Immunol 29 (5):633-9 (1992) with regard to ADCC sites inIgG1. These sites, as well, are not required for the fusion molecules ofthe present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the nativeFc may be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

The term “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means. As usedherein the term “multimer” as applied to Fc domains or moleculescomprising Fc domains refers to molecules having two or more polypeptidechains associated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizingsuch a native Fc. The term “dimer” as applied to Fc domains or moleculescomprising Fc domains refers to molecules having two polypeptide chainsassociated covalently or non-covalently.

Additionally, an alternative vehicle according to the present inventionis a non-Fc domain protein, polypeptide, peptide, antibody, antibodyfragment, or small molecule (e.g., a peptidomimetic compound) capable ofbinding to a salvage receptor. For example, one could use as a vehicle apolypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14,1998 to Presta et al. Peptides could also be selected by phage displayfor binding to the FcRn salvage receptor. Such salvage receptor-bindingcompounds are also included within the meaning of “vehicle”, and arewithin the scope of this invention. Such vehicles should be selected forincreased half-life (e.g., by avoiding sequences recognized byproteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

In addition, polymer vehicles may also be used to construct the bindingagents of the present invention. Various means for attaching chemicalmoieties useful as vehicles are currently available, see, e.g., PatentCooperation Treaty (“PCT”) International Publication No. WO 96/11953,entitled “N-Terminally Chemically Modified Protein Compositions andMethods,” herein incorporated by reference in its entirety. This PCTpublication discloses, among other things, the selective attachment ofwater soluble polymers to the N-terminus of proteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be linear or branched.The average molecular weight of the PEG will preferably range from about2 kDa to about 100 kDa, more preferably from about 5 kDa to about 50kDa, most preferably from about 5 kDa to about 10 kDa. The PEG groupswill generally be attached to the compounds of the invention viaacylation or reductive alkylation through a reactive group on the PEGmoiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactivegroup on the inventive compound (e.g., an aldehyde, amino, or estergroup). A useful strategy for the PEGylation of synthetic peptidesconsists of combining, through forming a conjugate linkage in solution,a peptide and a PEG moiety, each bearing a special functionality that ismutually reactive toward the other. The peptides can be easily preparedwith conventional solid phase synthesis as known in the art. Thepeptides are “preactivated” with an appropriate functional group at aspecific site. The precursors are purified and fully characterized priorto reacting with the PEG moiety. Ligation of the peptide with PEGusually takes place in aqueous phase and can be easily monitored byreverse phase analytical HPLC. The PEGylated peptides can be easilypurified by preparative HPLC and characterized by analytical HPLC, aminoacid analysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by a1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kDa to about 70 kDa. Dextran is a suitable water-solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference. Dextran of about 1 kDa to about 20 kDa is preferred whendextran is used as a vehicle in accordance with the present invention.

Linkers

The binding agents of the present invention may optionally furthercomprise a “linker” group. Linkers serve primarily as a spacer between apeptide and a vehicle or between two peptides of the binding agents ofthe present invention. In one embodiment, the linker is made up of aminoacids linked together by peptide bonds, preferably from 1 to 20 aminoacids linked by peptide bonds, wherein the amino acids are selected fromthe 20 naturally occurring amino acids. One or more of these amino acidsmay be glycosylated, as is understood by those in the art. In oneembodiment, the 1 to 20 amino acids are selected from glycine, alanine,proline, asparagine, glutamine, and lysine. Preferably, a linker is madeup of a majority of amino acids that are sterically unhindered, such asglycine and alanine. Thus, exemplary linkers are polyglycines(particularly (Gly)₅, (Gly)₈, poly(Gly-Ala), and polyalanines. As usedherein, the designation “g” refers to a glycine homopeptide linkers. Asshown in Table II, “gn” refers to a 5× gly linker at the N terminus,while “gc” refers to 5× gly linker at the C terminus Combinations of Glyand Ala are also preferred. One exemplary linker sequence useful forconstructing the binding agents of the present invention is thefollowing: gsgsatggsgstassgsgsatg (Seq ID No: 305). This linker sequenceis referred to as the “k” or 1k sequence. The designations “kc”, asfound in Table II, refers to the k linker at the C-terminus, while thedesignation “kn”, refers to the k linker at the N-terminus.

The linkers of the present invention may also be non-peptide linkers.For example, alkyl linkers such as —NH—(CH₂)s-C(O)—, wherein s=2-20 canbe used. These alkyl linkers may further be substituted by anynon-sterically hindering group such as lower alkyl (e.g., C₁-C₆) loweracyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplarynon-peptide linker is a PEG linker, and has a molecular weight of 100 to5000 kDa, preferably 100 to 500 kDa. The peptide linkers may be alteredto form derivatives in the same manner as above.

Exemplary Binding Agents

The binding agents of the present invention comprise at least onepeptide capable of binding myostatin. In one embodiment, the myostatinbinding peptide is between about 5 and about 50 amino acids in length,in another, between about 10 and 30 amino acids in length, and inanother, between about 10 and 25 amino acids in length. In oneembodiment the myostatin binding peptide comprises the amino acidsequence WMCPP (SEQ ID NO: 633). In other embodiment, the myostatinbinding peptide comprises the amino acid sequence Ca₁a₂ Wa₃ WMCPP (SEQID NO: 352), wherein a₁, a₂ and a₃ are selected from a neutralhydrophobic, neutral polar, or basic amino acid. In another embodimentthe myostatin binding peptide comprises the amino acid sequence Cb₁b₂Wb₃ WMCPP (SEQ ID NO: 353), wherein b₁ is selected from any one of theamino acids T, I, or R; b₂ is selected from any one of R, S, Q; b₃ isselected from any one of P, R and Q, and wherein the peptide is between10 and 50 amino acids in length, and physiologically acceptable saltsthereof.

In another embodiment, the myostatin binding peptide comprises theformula:c₁c₂c₃c₄c₅c₆ Cc₇c₈ Wc₉ WMCPPc₁₀c₁₁c₁₂c₁₃ (SEQ ID NO: 354),

wherein:

c₁ is absent or any amino acid;

c₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₄ is absent or any amino acid;

c₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

c₇ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₈ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₉ is a neutral hydrophobic, neutral polar or basic amino acid; and

c₁₀ to c₁₃ is any amino acid; and wherein the peptide is between 20 and50 amino acids in length, and physiologically acceptable salts thereof.

A related embodiment the myostatin binding peptide comprises theformula:d₁d₂d₃d₄d₅d₆ Cd₇d₈ Wd₉ WMCPPd₁₀d₁₁d₁₂d₁₃ (SEQ ID NO: 355),

wherein

d₁ is absent or any amino acid;

d₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₄ is absent or any amino acid;

d₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

d₇ is selected from any one of the amino acids T, I, or R;

d₈ is selected from any one of R, S, Q;

d₉ is selected from any one of P, R and Q, and

d₁₀ to d₁₃ is selected from any amino acid,

and wherein the peptide is between 20 and 50 amino acids in length, andphysiologically acceptable salts thereof.

Additional embodiments of binding agents comprise at least one of thefollowing peptides:

(1) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence WYe₁e₂ Ye₃ G, (SEQ ID NO: 356),

wherein e₁ is P, S or Y,

e₂ is C or Q, and

e₃ is G or H, wherein the peptide is between 7 and 50 amino acids inlength, and physiologically acceptable salts thereof;

(2) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence f₁ EMLf ₂ SLf₃f₄ LL, (SEQ ID NO: 455),

wherein f₁ is M or I,

f₂ is any amino acid,

f₃ is L or F,

f₄ is E, Q or D;

and wherein the peptide is between 7 and 50 amino acids in length, andphysiologically acceptable salts thereof;

(3) a peptide capable of binding myostatin wherein the peptide comprisesthe sequence Lg₁g₂ LLg₃g₄ L, (SEQ ID NO: 456), wherein

g₁ is Q, D or E,

g₂ is S, Q, D or E,

g₃ is any amino acid,

g₄ is L, W, F, or Y, and wherein the peptide is between 8 and 50 aminoacids in length, and physiologically acceptable salts thereof;

(4) a peptide capable of binding myostatin, wherein the peptidecomprises the sequence h₁h₂h₃h₄h₅h₆h₇h₈h₉ (SEQ ID NO: 457), wherein

h₁ is R or D,

h₂ is any amino acid,

h₃ is A, T S or Q,

h₄ is L or M,

h₅ is L or S,

h₆ is any amino acid,

h₇ is F or E,

h₈ is W, F or C,

h₉ is L, F, M or K, and wherein the peptide is between 9 and 50 aminoacids in length, and physiologically acceptable salts thereof.

In one embodiment, the binding agents of the present invention furthercomprise at least one vehicle such as a polymer or an Fc domain, and mayfurther comprise at least one linker sequence. In this embodiment, thebinding agents of the present invention are constructed so that at leastone myostatin-binding peptide is covalently attached to at least onevehicle. The peptide or peptides are attached directly or indirectlythrough a linker sequence, to the vehicle at the N-terminal, C-terminalor an amino acid sidechain of the peptide. In this embodiment, thebinding agents of the present invention have the following generalizedstructure:(X¹)_(a)—F¹—(X²)_(b),

or multimers thereof;

wherein F¹ is a vehicle; and X¹ and X² are each independently selectedfrom

-(L¹)_(c)-P¹;

-(L¹)_(c)-P¹-(L²)_(d)-P²;

-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³;

and -(L¹)_(e)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;

wherein P¹, P², P³, and P⁴ are peptides capable of binding myostatin;and

L¹, L², L³, and L⁴ are each linkers; and a, b, c, d, e, and f are eachindependently 0 or 1,

provided that at least one of a and b is 1.

In one embodiment of the binding agents having this generalizedstructure, the peptides P¹, P², P³, and P⁴ can be selected from one ormore of any of the peptides comprising the sequences provided above.Peptides P¹, P², P³, and P⁴ can be selected from one or more peptidescomprising any of the following sequences: SEQ ID NO: 633, SEQ ID NO:352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQID NO: 455, SEQ ID NO: 456, or SEQ ID NO: 457.

In a further embodiment, the vehicles of binding agents having thegeneral formula above are Fc domains. The peptides are therefore fusedto an Fc domain, either directly or indirectly, thereby providingpeptibodies. The peptibodies of the present invention display a highbinding affinity for myostatin and can inhibit the activity of myostatinas demonstrated by in vitro assays and in vivo testing in animalsprovided herein.

The present invention also provides nucleic acid molecules comprisingpolynucleotides encoding the peptides, peptibodies, and peptide andpeptibody variants and derivatives of the present invention. Exemplarynucleotides sequences are given below.

Variants and Derivatives of Peptides and Peptibodies

The binding agents of the present invention also encompass variants andderivatives of the peptides and peptibodies described herein. Since boththe peptides and peptibodies of the present invention can be describedin terms of their amino acid sequence, the terms “variants” and“derivatives” can be said to apply to a peptide alone, or a peptide as acomponent of a peptibody. As used herein, the term “peptide variants”refers to peptides or peptibodies having one or more amino acid residuesinserted, deleted or substituted into the original amino acid sequenceand which retain the ability to bind to myostatin and modify itsactivity. As used herein, fragments of the peptides or peptibodies areincluded within the definition of “variants”.

It is understood that any given peptide or peptibody may contain one ortwo or all three types of variants. Insertional and substitutionalvariants may contain natural amino acids, as well as non-naturallyoccurring amino acids or both.

Peptide and peptibody variants also include mature peptides andpeptibodies wherein leader or signal sequences are removed, and theresulting proteins having additional amino terminal residues, whichamino acids may be natural or non-natural. Peptibodies with anadditional methionyl residue at amino acid position −1 (Met⁻¹-peptibody)are contemplated, as are peptibodies with additional methionine andlysine residues at positions −2 and −1 (Met⁻²-Lys⁻¹-). Variants havingadditional Met, Met-Lys, Lys residues (or one or more basic residues, ingeneral) are particularly useful for enhanced recombinant proteinproduction in bacterial host cells.

Peptide or peptibody variants of the present invention also includespeptides having additional amino acid residues that arise from use ofspecific expression systems. For example, use of commercially availablevectors that express a desired polypeptide as part ofglutathione-S-transferase (GST) fusion product provides the desiredpolypeptide having an additional glycine residue at amino acidposition-1 after cleavage of the GST component from the desiredpolypeptide. Variants which result from expression in other vectorsystems are also contemplated, including those wherein histidine tagsare incorporated into the amino acid sequence, generally at the carboxyand/or amino terminus of the sequence.

In one example, insertional variants are provided wherein one or moreamino acid residues, either naturally occurring or non-naturallyoccurring amino acids, are added to a peptide amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the peptibody amino acidsequence. Insertional variants with additional residues at either orboth termini can include, for example, fusion proteins and proteinsincluding amino acid tags or labels. Insertional variants includepeptides in which one or more amino acid residues are added to thepeptide amino acid sequence or fragment thereof.

Insertional variants also include fusion proteins wherein the aminoand/or carboxy termini of the peptide or peptibody is fused to anotherpolypeptide, a fragment thereof or amino acids which are not generallyrecognized to be part of any specific protein sequence. Examples of suchfusion proteins are immunogenic polypeptides, proteins with longcirculating half lives, such as immunoglobulin constant regions, markerproteins, proteins or polypeptides that facilitate purification of thedesired peptide or peptibody, and polypeptide sequences that promoteformation of multimeric proteins (such as leucine zipper motifs that areuseful in dimer formation/stability).

This type of insertional variant generally has all or a substantialportion of the native molecule, linked at the N- or C-terminus, to allor a portion of a second polypeptide. For example, fusion proteinstypically employ leader sequences from other species to permit therecombinant expression of a protein in a heterologous host. Anotheruseful fusion protein includes the addition of an immunologically activedomain, such as an antibody epitope, to facilitate purification of thefusion protein. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include but are not limited to the glutathione-S-transferase(GST) system (Pharmacia), the maltose binding protein system (NEB,Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×Hissystem (Qiagen, Chatsworth, Calif.). These systems are capable ofproducing recombinant peptides and/or peptibodies bearing only a smallnumber of additional amino acids, which are unlikely to significantlyaffect the activity of the peptide or peptibody. For example, both theFLAG system and the 6×His system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of a polypeptide to its native conformation. Another N-terminalfusion that is contemplated to be useful is the fusion of a Met-Lysdipeptide at the N-terminal region of the protein or peptides. Such afusion may produce beneficial increases in protein expression oractivity.

Other fusion systems produce polypeptide hybrids where it is desirableto excise the fusion partner from the desired peptide or peptibody. Inone embodiment, the fusion partner is linked to the recombinantpeptibody by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

The invention also provides fusion polypeptides which comprise all orpart of a peptide or peptibody of the present invention, in combinationwith truncated tissue factor (tTF). tTF is a vascular targeting agentconsisting of a truncated form of a human coagulation-inducing proteinthat acts as a tumor blood vessel clotting agent, as described U.S. Pat.Nos. 5,877,289; 6,004,555; 6,132,729; 6,132,730; 6,156,321; and EuropeanPatent No. EP 0988056. The fusion of tTF to the anti-myostatin peptibodyor peptide, or fragments thereof facilitates the delivery ofanti-myostatin antagonists to target cells, for example, skeletal musclecells, cardiac muscle cells, fibroblasts, pre-adipocytes, and possiblyadipocytes.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a peptide or peptibody are removed.Deletions can be effected at one or both termini of the peptibody, orfrom removal of one or more residues within the peptibody amino acidsequence. Deletion variants necessarily include all fragments of apeptide or peptibody.

In still another aspect, the invention provides substitution variants ofpeptides and peptibodies of the invention. Substitution variants includethose peptides and peptibodies wherein one or more amino acid residuesare removed and replaced with one or more alternative amino acids, whichamino acids may be naturally occurring or non-naturally occurring.Substitutional variants generate peptides or peptibodies that are“similar” to the original peptide or peptibody, in that the twomolecules have a certain percentage of amino acids that are identical.Substitution variants include substitutions of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, and 20 amino acids within a peptide or peptibody, wherein thenumber of substitutions may be up to ten percent of the amino acids ofthe peptide or peptibody. In one aspect, the substitutions areconservative in nature, however, the invention embraces substitutionsthat are also non-conservative and also includes unconventional aminoacids.

Identity and similarity of related peptides and peptibodies can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press (1987);Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York (1991); and Carillo et al., SIAM J. AppliedMath., 48:1073 (1988).

Preferred methods to determine the relatedness or percent identity oftwo peptides or polypeptides, or a polypeptide and a peptide, aredesigned to give the largest match between the sequences tested. Methodsto determine identity are described in publicly available computerprograms. Preferred computer program methods to determine identitybetween two sequences include, but are not limited to, the GCG programpackage, including GAP (Devereux et al., Nucl. Acid. Res., 12:387(1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.,BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410(1990)). The BLASTX program is publicly available from the NationalCenter for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul etal., supra (1990)). The well-known Smith Waterman algorithm may also beused to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod will result in an alignment that spans at least ten percent ofthe full length of the target polypeptide being compared, i.e., at least40 contiguous amino acids where sequences of at least 400 amino acidsare being compared, 30 contiguous amino acids where sequences of atleast 300 to about 400 amino acids are being compared, at least 20contiguous amino acids where sequences of 200 to about 300 amino acidsare being compared, and at least 10 contiguous amino acids wheresequences of about 100 to 200 amino acids are being compared. Forexample, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is typically calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., Atlas of ProteinSequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix;Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 (1992) forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, for example, the parameters for a polypeptidesequence comparison can be made with the following: Algorithm: Needlemanet al., J. Mol. Biol., 48:443-453 (1970); Comparison matrix: BLOSUM 62from Henikoff et al., supra (1992); Gap Penalty: 12; Gap Length Penalty:4; Threshold of Similarity: 0, along with no penalty for end gaps.

In certain embodiments, the parameters for polynucleotide moleculesequence (as opposed to an amino acid sequence) comparisons can be madewith the following: Algorithm: Needleman et al., supra (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50: Gap LengthPenalty: 3

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

Stereoisomers (e.g., D-amino acids) of the twenty conventional(naturally occurring) amino acids, non-naturally occurring amino acidssuch as α-, α-disubstituted amino acids, N-alkyl amino acids, lacticacid, and other unconventional amino acids may also be suitablecomponents for peptides of the present invention. Examples ofnon-naturally occurring amino acids include, for example: aminoadipicacid, beta-alanine, beta-aminopropionic acid, aminobutyric acid,piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine,diaminopimelic acid, diaminopropionic acid, N-ethylglycine,N-ethylasparagine, hyroxylysine, all0-hydroxylysine, hydroxyproline,isodesmosine, allo-isoleucine, N-methylglycine, sarcosine,N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine,4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,8-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and amino acids (e.g., 4-hydroxyproline).

Naturally occurring residues may be divided into (overlapping) classesbased on common side chain properties:

-   -   1) neutral hydrophobic: Met, Ala, Val, Leu, Ile, Pro, Trp, Met,        Phe;    -   2) neutral polar: Cys, Ser, Thr, Asn, Gln, Tyr, Gly;    -   3) acidic: Asp, Glu;    -   4) basic: H is, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Substitutions of amino acids may be conservative, which producespeptides having functional and chemical characteristics similar to thoseof the original peptide. Conservative amino acid substitutions involveexchanging a member of one of the above classes for another member ofthe same class. Conservative changes may encompass unconventional aminoacid residues, which are typically incorporated by chemical peptidesynthesis rather than by synthesis in biological systems. These includepeptidomimetics and other reversed or inverted forms of amino acidmoieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. These changes canresult in substantial modification in the functional and/or chemicalcharacteristics of the peptides. In making such changes, according tocertain embodiments, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional peptibody or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1 below.

Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln, Glu, Asp Gln Asp Glu, Gln, Asp Glu Cys Ser, Ala Ser GlnAsn, Glu, Asp Asn Glu Asp, Gln, Asn Asp Gly Pro, Ala Ala HisAsn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine LeuNorleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino- Argbutyric Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, TyrLeu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

One skilled in the art will be able to produce variants of the peptidesand peptibodies of the present invention by random substitution, forexample, and testing the resulting peptide or peptibody for bindingactivity using the assays described herein.

Additionally, one skilled in the art can review structure-functionstudies or three-dimensional structural analysis in order to identifyresidues in similar polypeptides that are important for activity orstructure. In view of such a comparison, one can predict the importanceof amino acid residues in a protein that correspond to amino acidresidues which are important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues. Thevariants can then be screened using activity assays as described herein.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13 (2): 222-245 (1974);Chou et al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a protein's structure. See Holm et al., Nucl.Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al.,Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limitednumber of folds in a given protein and that once a critical number ofstructures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, peptide or peptibody variants includeglycosylation variants wherein one or more glycosylation sites such as aN-linked glycosylation site, has been added to the peptibody. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution or addition of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions which eliminate this sequence will remove an existingN-linked carbohydrate chain. Also provided is a rearrangement ofN-linked carbohydrate chains wherein one or more N-linked glycosylationsites (typically those that are naturally occurring) are eliminated andone or more new N-linked sites are created.

The invention also provides “derivatives” of the peptides or peptibodiesof the present invention. As used herein the term “derivative” refers tomodifications other than, or in addition to, insertions, deletions, orsubstitutions of amino acid residues which retain the ability to bind tomyostatin.

Preferably, the modifications made to the peptides of the presentinvention to produce derivatives are covalent in nature, and include forexample, chemical bonding with polymers, lipids, other organic, andinorganic moieties. Derivatives of the invention may be prepared toincrease circulating half-life of a peptibody, or may be designed toimprove targeting capacity for the peptibody to desired cells, tissues,or organs.

The invention further embraces derivative binding agents covalentlymodified to include one or more water soluble polymer attachments, suchas polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol,as described U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192; and 4,179,337. Still other useful polymers known in the artinclude monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of these polymers. Particularlypreferred are peptibodies covalently modified with polyethylene glycol(PEG) subunits. Water-soluble polymers may be bonded at specificpositions, for example at the amino terminus of the peptibodies, orrandomly attached to one or more side chains of the polypeptide. The useof PEG for improving the therapeutic capacity for binding agents, e.g.peptibodies, and for humanized antibodies in particular, is described inU.S. Pat. No. 6,133,426 to Gonzales et al., issued Oct. 17, 2000.

The invention also contemplates derivatizing the peptide and/or vehicleportion of the myostatin binding agents. Such derivatives may improvethe solubility, absorption, biological half-life, and the like of thecompounds. The moieties may alternatively eliminate or attenuate anyundesirable side-effect of the compounds and the like. Exemplaryderivatives include compounds in which:

1. The derivative or some portion thereof is cyclic. For example, thepeptide portion may be modified to contain two or more Cys residues(e.g., in the linker), which could cyclize by disulfide bond formation.

2. The derivative is cross-linked or is rendered capable ofcross-linking between molecules. For example, the peptide portion may bemodified to contain one Cys residue and thereby be able to form anintermolecular disulfide bond with a like molecule. The derivative mayalso be cross-linked through its C-terminus.

3. One or more peptidyl [—C(O)NR-] linkages (bonds) is replaced by anon-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂-carbamate[—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea[—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR₆—wherein R₆ is lower alkyl].

4. The N-terminus is derivatized. Typically, the N-terminus may beacylated or modified to a substituted amine. Exemplary N-terminalderivative groups include —NRR₁ (other than —NH₂), —NRC(O)R₁,—NRC(O)ORi, —NRS(O)₂R₁, —NHC(O)NHR₁, succinimide, orbenzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R1 are each independentlyhydrogen or lower alkyl and wherein the phenyl ring may be substitutedwith 1 to 3 substituents selected from the group consisting of C₁-C₄alkyl, C₁-C₄ alkoxy, chloro, and bromo.

5. The free C-terminus is derivatized. Typically, the C-terminus isesterified or amidated. For example, one may use methods described inthe art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention at theC-terminus. Likewise, one may use methods described in the art to add—NH₂, (or “capping” with an —NH₂ group) to compounds of this inventionat the C-terminus. Exemplary C-terminal derivative groups include, forexample, —C(O)R₂ wherein R₂ is lower alkoxy or —NR₃, R₄ wherein R₃ andR₄ are independently hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).

6. A disulfide bond is replaced with another, preferably more stable,cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al., JMed Chem 39: 3814-9 (1996), Alberts et al., Thirteenth Am Pep Symp,357-9 (1993).

7. One or more individual amino acid residues is modified. Variousderivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl side chain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al., (supra).

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithiol]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains [see,for example, Creighton, Proteins: Structure and Molecule Properties(W.H. Freeman & Co., San Francisco), pp. 79-86 (1983)].

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Additional derivatives include non-peptide analogs that provide astabilized structure or lessened biodegradation, are also contemplated.Peptide mimetic analogs can be prepared based on a selected inhibitorypeptide by replacement of one or more residues by nonpeptide moieties.Preferably, the nonpeptide moieties permit the peptide to retain itsnatural confirmation, or stabilize a preferred, e.g., bioactive,confirmation which retains the ability to recognize and bind myostatin.In one aspect, the resulting analog/mimetic exhibits increased bindingaffinity for myostatin. One example of methods for preparation ofnonpeptide mimetic analogs from peptides is described in Nachman et al.,Regul Pept 57:359-370 (1995). If desired, the peptides of the inventioncan be modified, for instance, by glycosylation, amidation,carboxylation, or phosphorylation, or by the creation of acid additionsalts, amides, esters, in particular C-terminal esters, and N-acylderivatives of the peptides of the invention. The peptibodies also canbe modified to create peptide derivatives by forming covalent ornoncovalent complexes with other moieties. Covalently-bound complexescan be prepared by linking the chemical moieties to functional groups onthe side chains of amino acids comprising the peptibodies, or at the N-or C-terminus.

In particular, it is anticipated that the peptides can be conjugated toa reporter group, including, but not limited to a radiolabel, afluorescent label, an enzyme (e.g., that catalyzes a colorimetric orfluorometric reaction), a substrate, a solid matrix, or a carrier (e.g.,biotin or avidin). The invention accordingly provides a moleculecomprising a peptibody molecule, wherein the molecule preferably furthercomprises a reporter group selected from the group consisting of aradiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix,and a carrier. Such labels are well known to those of skill in the art,e.g., biotin labels are particularly contemplated. The use of suchlabels is well known to those of skill in the art and is described in,e.g., U.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; and 4,277,437.Other labels that will be useful include but are not limited toradioactive labels, fluorescent labels and chemiluminescent labels. U.S.patents concerning use of such labels include, for example, U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; and 3,996,345. Any of thepeptibodies of the present invention may comprise one, two, or more ofany of these labels.

Methods of Making Peptides and Peptibodies

The peptides of the present invention can be generated using a widevariety of techniques known in the art. For example, such peptides canbe synthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young (supra); Tam et al., J. Am Chem Soc,105:6442, (1983); Merrifield, Science 232:341-347 (1986); Barany andMerrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, NewYork, 1-284; Barany et al., Int J Pep Protein Res, 30:705-739 (1987);and U.S. Pat. No. 5,424,398, each incorporated herein by reference.

Solid phase peptide synthesis methods use acopoly(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer.These methods for peptide synthesis use butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxy-carbonyl(FMOC) protection of alpha-amino groups.Both methods involve stepwise syntheses whereby a single amino acid isadded at each step starting from the C-terminus of the peptide (See,Coligan et al., Curr Prot Immunol, Wiley Interscience, 1991, Unit 9). Oncompletion of chemical synthesis, the synthetic peptide can bedeprotected to remove the t-BOC or FMOC amino acid blocking groups andcleaved from the polymer by treatment with acid at reduced temperature(e.g., liquid HF-10% anisole for about 0.25 to about 1 hours at 0° C.).After evaporation of the reagents, the peptides are extracted from thepolymer with 1% acetic acid solution that is then lyophilized to yieldthe crude material. This can normally be purified by such techniques asgel filtration on Sephadex G-15 using 5% acetic acid as a solvent.Lyophilization of appropriate fractions of the column will yield thehomogeneous peptides or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by the solid phase Edman degradation.

Phage display techniques can be particularly effective in identifyingthe peptides of the present invention as described above. Briefly, aphage library is prepared (using e.g. ml 13, fd, or lambda phage),displaying inserts from 4 to about 80 amino acid residues. The insertsmay represent, for example, a completely degenerate or biased array.Phage-bearing inserts that bind to the desired antigen are selected andthis process repeated through several cycles of reselection of phagethat bind to the desired antigen. DNA sequencing is conducted toidentify the sequences of the expressed peptides. The minimal linearportion of the sequence that binds to the desired antigen can bedetermined in this way. The procedure can be repeated using a biasedlibrary containing inserts containing part or all of the minimal linearportion plus one or more additional degenerate residues upstream ordownstream thereof. These techniques may identify peptides of theinvention with still greater binding affinity for myostatin than agentsalready identified herein.

Regardless of the manner in which the peptides are prepared, a nucleicacid molecule encoding each such peptide can be generated using standardrecombinant DNA procedures. The nucleotide sequence of such moleculescan be manipulated as appropriate without changing the amino acidsequence they encode to account for the degeneracy of the nucleic acidcode as well as to account for codon preference in particular hostcells.

The present invention also provides nucleic acid molecules comprisingpolynucleotide sequences encoding the peptides and peptibodies of thepresent invention. These nucleic acid molecules include vectors andconstructs containing polynucleotides encoding the peptides andpeptibodies of the present invention, as well as peptide and peptibodyvariants and derivatives. Exemplary nucleic acid molecules are providedin the Examples below.

Recombinant DNA techniques also provide a convenient method forpreparing full length peptibodies and other large polypeptide bindingagents of the present invention, or fragments thereof A polynucleotideencoding the peptibody or fragment may be inserted into an expressionvector, which can in turn be inserted into a host cell for production ofthe binding agents of the present invention. Preparation of exemplarypeptibodies of the present invention are described in Example 2 below.

A variety of expression vector/host systems may be utilized to expressthe peptides and peptibodies of the invention. These systems include butare not limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or animalcell systems. One preferred host cell line is E. coli strain 2596 (ATCC#202174), used for expression of peptibodies as described below inExample 2. Mammalian cells that are useful in recombinant proteinproductions include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138,BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.

The term “expression vector” refers to a plasmid, phage, virus orvector, for expressing a polypeptide from a polynucleotide sequence. Anexpression vector can comprise a transcriptional unit comprising anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, promoters or enhancers, (2) astructural or sequence that encodes the binding agent which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant protein isexpressed without a leader or transport sequence, it may include anamino terminal methionyl residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal peptide product.

For example, the peptides and peptibodies may be recombinantly expressedin yeast using a commercially available expression system, e.g., thePichia Expression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol. The secreted peptide is purified from the yeast growthmedium using the methods used to purify the peptide from bacterial andmammalian cell supernatants.

Alternatively, the cDNA encoding the peptide and peptibodies may becloned into the baculovirus expression vector pVL1393 (PharMingen, SanDiego, Calif.). This vector can be used according to the manufacturer'sdirections (PharMingen) to infect Spodoptera frugiperda cells in sF9protein-free media and to produce recombinant protein. The recombinantprotein can be purified and concentrated from the media using aheparin-Sepharose column (Pharmacia).

Alternatively, the peptide or peptibody may be expressed in an insectsystem. Insect systems for protein expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) can be used as a vector to express foreigngenes in Spodoptera frugiperda cells or in Trichoplusia larvae. Thepeptide coding sequence can be cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the peptide will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses can be used to infect S.frugiperda cells or Trichoplusia larvae in which the peptide isexpressed (Smith et al., J Virol 46: 584 (1983); Engelhard et al., ProcNat Acad Sci (USA) 91: 3224-7 (1994)).

In another example, the DNA sequence encoding the peptide can beamplified by PCR and cloned into an appropriate vector for example,pGEX-3X (Pharmacia). The pGEX vector is designed to produce a fusionprotein comprising glutathione-S-transferase (GST), encoded by thevector, and a protein encoded by a DNA fragment inserted into thevector's cloning site. The primers for PCR can be generated to includefor example, an appropriate cleavage site. Where the fusion moiety isused solely to facilitate expression or is otherwise not desirable as anattachment to the peptide of interest, the recombinant fusion proteinmay then be cleaved from the GST portion of the fusion protein. ThepGEX-3X/specific binding agent peptide construct is transformed into E.coli XL-1 Blue cells (Stratagene, La Jolla Calif.), and individualtransformants isolated and grown. Plasmid DNA from individualtransformants can be purified and partially sequenced using an automatedsequencer to confirm the presence of the desired specific binding agentencoding nucleic acid insert in the proper orientation.

The fusion protein, which may be produced as an insoluble inclusion bodyin the bacteria, can be purified as follows. Host cells are collected bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15 minutesat room temperature. The lysate can be cleared by sonication, and celldebris can be pelleted by centrifugation for 10 minutes at 12,000×g. Thefusion protein-containing pellet can be resuspended in 50 mM Tris, pH 8,and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.at 6000×g. The pellet can be resuspended in standard phosphate bufferedsaline solution (PBS) free of Mg++ and Ca++. The fusion protein can befurther purified by fractionating the resuspended pellet in a denaturingSDS-PAGE (Sambrook et al., supra). The gel can be soaked in 0.4 M KCl tovisualize the protein, which can be excised and electroeluted ingel-running buffer lacking SDS. If the GST/fusion protein is produced inbacteria as a soluble protein, it can be purified using the GSTPurification Module (Pharmacia).

The fusion protein may be subjected to digestion to cleave the GST fromthe peptide of the invention. The digestion reaction (20-40 mg fusionprotein, 20-30 units human thrombin (4000 U/mg, Sigma) in 0.5 ml PBS canbe incubated 16-48 hrs at room temperature and loaded on a denaturingSDS-PAGE gel to fractionate the reaction products. The gel can be soakedin 0.4 M KCl to visualize the protein bands. The identity of the proteinband corresponding to the expected molecular weight of the peptide canbe confirmed by amino acid sequence analysis using an automatedsequencer (Applied Biosystems Model 473A, Foster City, Calif.).Alternatively, the identity can be confirmed by performing HPLC and/ormass spectometry of the peptides.

Alternatively, a DNA sequence encoding the peptide can be cloned into aplasmid containing a desired promoter and, optionally, a leader sequence(Better et al., Science 240:1041-43 (1988)). The sequence of thisconstruct can be confirmed by automated sequencing. The plasmid can thenbe transformed into E. coli strain MC1061 using standard proceduresemploying CaCl2 incubation and heat shock treatment of the bacteria(Sambrook et al., supra). The transformed bacteria can be grown in LBmedium supplemented with carbenicillin, and production of the expressedprotein can be induced by growth in a suitable medium. If present, theleader sequence can effect secretion of the peptide and be cleavedduring secretion.

Mammalian host systems for the expression of recombinant peptides andpeptibodies are well known to those of skill in the art. Host cellstrains can be chosen for a particular ability to process the expressedprotein or produce certain post-translation modifications that will beuseful in providing protein activity. Such modifications of the proteininclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation. Different hostcells such as CHO, HeLa, MDCK, 293, W138, and the like have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and can be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

It is preferable that transformed cells be used for long-term,high-yield protein production. Once such cells are transformed withvectors that contain selectable markers as well as the desiredexpression cassette, the cells can be allowed to grow for 1-2 days in anenriched media before they are switched to selective media. Theselectable marker is designed to allow growth and recovery of cells thatsuccessfully express the introduced sequences. Resistant clumps ofstably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell line employed.

A number of selection systems can be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr which confers resistance to methotrexate; gptwhich confers resistance to mycophenolic acid; neo which confersresistance to the aminoglycoside G418 and confers resistance tochlorsulfuron; and hygro which confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Purification and Refolding of Binding Agents

In some cases, the binding agents such as the peptides and/orpeptibodies of this invention may need to be “refolded” and oxidizedinto a proper tertiary structure and disulfide linkages generated inorder to be biologically active. Refolding can be accomplished using anumber of procedures well known in the art. Such methods include, forexample, exposing the solubilized polypeptide agent to a pH usuallyabove 7 in the presence of a chaotropic agent. The selection ofchaotrope is similar to the choices used for inclusion bodysolubilization, however a chaotrope is typically used at a lowerconcentration. Exemplary chaotropic agents are guanidine and urea. Inmost cases, the refolding/oxidation solution will also contain areducing agent plus its oxidized form in a specific ratio to generate aparticular redox potential which allows for disulfide shuffling to occurfor the formation of cysteine bridges. Some commonly used redox couplesinclude cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride,dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME.In many instances, a co-solvent may be used to increase the efficiencyof the refolding. Commonly used cosolvents include glycerol,polyethylene glycol of various molecular weights, and arginine.

It may be desirable to purify the peptides and peptibodies of thepresent invention. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the proteinaceous and non-proteinaceousfractions. Having separated the peptide and/or peptibody from otherproteins, the peptide or polypeptide of interest can be further purifiedusing chromatographic and electrophoretic techniques to achieve partialor complete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of peptibodies andpeptides or the present invention are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of a peptibodyor peptide of the present invention. The term “purified peptibody orpeptide” as used herein, is intended to refer to a composition,isolatable from other components, wherein the peptibody or peptide ispurified to any degree relative to its naturally-obtainable state. Apurified peptide or peptibody therefore also refers to a peptibody orpeptide that is free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a peptide or peptibody compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a peptide or peptibody composition inwhich the peptibody or peptide forms the major component of thecomposition, such as constituting about 50%, about 60%, about 70%, about80%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of thepeptide or peptibody will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific binding activity of an active fraction, or assessing the amountof peptide or peptibody within a fraction by SDS/PAGE analysis. Apreferred method for assessing the purity of a peptide or peptibodyfraction is to calculate the binding activity of the fraction, tocompare it to the binding activity of the initial extract, and to thuscalculate the degree of purification, herein assessed by a “-foldpurification number.” The actual units used to represent the amount ofbinding activity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not thepeptibody or peptide exhibits a detectable binding activity.

Various techniques suitable for use in purification will be well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies (immunoprecipitation) and thelike or by heat denaturation, followed by centrifugation; chromatographysteps such as affinity chromatography (e.g., Protein-A-Sepharose), ionexchange, gel filtration, reverse phase, hydroxylapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified binding agent.

There is no general requirement that the binding agents of the presentinvention always be provided in their most purified state. Indeed, it iscontemplated that less substantially purified binding agent productswill have utility in certain embodiments. Partial purification may beaccomplished by using fewer purification steps in combination, or byutilizing different forms of the same general purification scheme. Forexample, it is appreciated that a cation-exchange column chromatographyperformed utilizing an HPLC apparatus will generally result in a greater“-fold” purification than the same technique utilizing a low-pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of the peptide orpeptibody, or in maintaining binding activity of the peptide orpeptibody.

It is known that the migration of a peptide or polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE (Capaldiet al., Biochem Biophys Res Comm, 76: 425 (1977)). It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified bindingagent expression products may vary.

Activity of Myostatin Binding Agents

After the construction of the binding agents of the present invention,they are tested for their ability to bind myostatin and inhibit or blockmyostatin activity. Any number of assays or animal tests may be used todetermine the ability of the agent to inhibit or block myostatinactivity. Several assays used for characterizing the peptides andpeptibodies of the present invention are described in the Examplesbelow. One assay is the C2C12 pMARE-luc assay which makes use of amyostatin-responsive cell line (C2C12 myo blasts) transfected with aluciferase reporter vector containing myostatin/activin responseelements (MARE). Exemplary peptibodies are assayed by pre-incubating aseries of peptibody dilutions with myostatin, and then exposing thecells to the incubation mixture. The resulting luciferase activity isdetermined, and a titration curve is generated from the series ofpeptibody dilutions. The IC₅₀ (the concentration of peptibody to achieve50% inhibition of myostatin activity as measured by luciferase activity)was then determined A second assay described below is a BIAcore® assayto determine the kinetic parameters k_(a) (association rate constant),k_(d) (dissociation rate constant), and K_(D) (dissociation equilibriumconstant) for the myostatin binding agents. Lower dissociationequilibrium constants (K_(D), expressed in nM) indicated a greateraffinity of the peptibody for myostatin. Additional assays includeblocking assays, to determine whether a binding agent such as apeptibody is neutralizing (prevents binding of myostatin to itsreceptor), or non-neutralizing (does not prevent binding of myostatin toits receptor); selectivity assays, which determine if the binding agentsof the present invention bind selectively to myostatin and not to otherTGFβ family members; and KinEx A™ assays or solution-based equilibriumassays, which also determine K_(D) and are considered to be moresensitive in some circumstances. These assays are described in Example3.

FIG. 1 shows the IC₅₀ of a peptide compared with the IC₅₀ of thepeptibody form of the peptide. This demonstrates that the peptibody issignificantly more effective at inhibiting myostatin activity than thepeptide alone. In addition, affinity-matured peptibodies generallyexhibit improved IC₅₀ and K_(D) values compared with the parent peptidesand peptibodies. The IC₅₀ values for a number of exemplary affinitymatured peptibodies are shown in Table VII, Example 7 below.Additionally, in some instances, making a 2× version of a peptibody,where two peptides are attached in tandem, increase the activity of thepeptibody both in vitro and in vivo.

In vivo activities are demonstrated in the Examples below. Theactivities of the binding agents include anabolic activity increasinglean muscle mass in animal models, as well as decreasing the fat masswith respect to total body weight in treated animal models, andincreasing muscular strength in animal models.

Uses of the Myostatin Binding Agents

The myostatin binding agents of the present invention bind to myostatinand block or inhibit myostatin signaling within targeted cells. Thepresent invention provides methods and reagents for reducing the amountor activity of myostatin in an animal by administering an effectivedosage of one or more myostatin binding agents to the animal. In oneaspect, the present invention provides methods and reagents for treatingmyostatin-related disorders in an animal comprising administering aneffective dosage of one or more binding agents to the animal. Thesemyostatin-related disorders include but are not limited to various formsof muscle wasting, as well as metabolic disorders such as diabetes andrelated disorders, and bone degenerative diseases such as osteoporosis.

As shown in the Example 8 below, exemplary peptibodies of the presentinvention dramatically increases lean muscle mass in the CD1 nu/nu mousemodel. This in vivo activity correlates to the in vitro binding andinhibitory activity described below for the same peptibodies.

Muscle wasting disorders include dystrophies such as Duchenne's musculardystrophy, progressive muscular dystrophy, Becker's type musculardystrophy, Dejerine-Landouzy muscular dystrophy, Erb's musculardystrophy, and infantile neuroaxonal muscular dystrophy. For example,blocking myostatin through use of antibodies in vivo improved thedystrophic phenotype of the mdxmouse model of Duchenne musculardystrophy (Bogdanovich et al, Nature 420, 28 (2002)). The peptibodies ofthe present invention increase lean muscle mass as a percentage of bodyweight and decrease fat mass as percentage of body weight whenadministered to an aged mdxmouse model.

Additional muscle wasting disorders arise from chronic disease such asamyotrophic lateral sclerosis, congestive obstructive pulmonary disease,cancer, AIDS, renal failure, and rheumatoid arthritis. For example,cachexia or muscle wasting and loss of body weight was induced inathymic nude mice by a systemically administered myostatin (Zimmers etal., supra). In another example, serum and intramuscular concentrationsof myostatin-immunoreactive protein was found to be increased in menexhibiting AIDS-related muscle wasting and was inversely related tofat-free mass (Gonzalez-Cadavid et al., PNAS USA 95: 14938-14943(1998)). Additional conditions resulting in muscle wasting may arisefrom inactivity due to disability such as confinement in a wheelchair,prolonged bedrest due to stroke, illness, spinal chord injury, bonefracture or trauma, and muscular atrophy in a microgravity environment(space flight). For example, plasma myostatin immunoreactive protein wasfound to increase after prolonged bedrest (Zachwieja et al. J GravitPhysiol. 6(2):11 (1999). It was also found that the muscles of ratsexposed to a microgravity environment during a space shuttle flightexpressed an increased amount of myostatin compared with the muscles ofrats which were not exposed (Lalani et al., J. Endocrin 167 (3):417-28(2000)).

In addition, age-related increases in fat to muscle ratios, andage-related muscular atrophy appear to be related to myostatin. Forexample, the average serum myostatin-immunoreactive protein increasedwith age in groups of young (19-35 yr old), middle-aged (36-75 yr old),and elderly (76-92 yr old) men and women, while the average muscle massand fat-free mass declined with age in these groups (Yarasheski et al. JNutr Aging 6(5):343-8 (2002)). It has also been shown that myostatingene knockout in mice increased myogenesis and decreased adipogenesis(Lin et al., Biochem Biophys Res Commun 291(3):701-6 (2002), resultingin adults with increased muscle mass and decreased fat accumulation andleptin secretion. Exemplary peptibodies improve the lean muscle mass tofat ratio in aged mdx mice as shown below.

In addition, myostatin has now been found to be expressed at low levelsin heart muscle and expression is upregulated in cardiomyocytes afterinfarct (Sharma et al., J Cell Physiol. 180 (1):1-9 (1999)). Therefore,reducing myostatin levels in the heart muscle may improve recovery ofheart muscle after infarct.

Myostatin also appears to influence metabolic disorders including type 2diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, andobesity. For example, lack of myostatin has been shown to improve theobese and diabetic phenotypes of two mouse models (Yen et al. supra). Ithas been demonstrated in the Examples below that decreasing myostatinactivity by administering the inhibitors of the present invention willdecrease the fat to muscle ratio in an animal, including aged animalmodels. Therefore, decreasing fat composition by administering theinhibitors of the present invention will improve diabetes, obesity, andhyperglycemic conditions in animals.

In addition, increasing muscle mass by reducing myostatin levels mayimprove bone strength and reduce osteoporosis and other degenerativebone diseases. It has been found, for example, that myostatin-deficientmice showed increased mineral content and density of the mouse humerusand increased mineral content of both trabecular and cortical bone atthe regions where the muscles attach, as well as increased muscle mass(Hamrick et al. Calcif Tissue Int 71(1):63-8 (2002)).

The present invention also provides methods and reagents for increasingmuscle mass in food animals by administering an effective dosage of themyostatin binding agent to the animal Since the mature C-terminalmyostatin polypeptide is identical in all species tested, myostatinbinding agents would be expected to be effective for increasing musclemass and reducing fat in any agriculturally important species includingcattle, chicken, turkeys, and pigs.

The binding agents of the present invention may be used alone or incombination with other therapeutic agents to enhance their therapeuticeffects or decrease potential side effects. The binding agents of thepresent invention possess one or more desirable but unexpectedcombination of properties to improve the therapeutic value of theagents. These properties include increased activity, increasedsolubility, reduced degradation, increased half-life, reduced toxicity,and reduced immunogenicity. Thus the binding agents of the presentinvention are useful for extended treatment regimes. In addition, theproperties of hydrophilicity and hydrophobicity of the compounds of theinvention are well balanced, thereby enhancing their utility for both invitro and especially in vivo uses. Specifically, compounds of theinvention have an appropriate degree of solubility in aqueous media thatpermits absorption and bioavailability in the body, while also having adegree of solubility in lipids that permits the compounds to traversethe cell membrane to a putative site of action, such as a particularmuscle mass.

The binding agents of the present invention are useful for treating a“subject” or any animal, including humans, when administered in aneffective dosage in a suitable composition.

In addition, the mystatin binding agents of the present invention areuseful for detecting and quantitating myostatin in a number of assays.These assays are described in more detail below.

In general, the binding agents of the present invention are useful ascapture agents to bind and immobilize myostatin in a variety of assays,similar to those described, for example, in Asai, ed., Methods in CellBiology, 37, Antibodies in Cell Biology, Academic Press, Inc., New York(1993). The binding agent may be labeled in some manner or may reactwith a third molecule such as an anti-binding agent antibody which islabeled to enable myostatin to be detected and quantitated. For example,a binding agent or a third molecule can be modified with a detectablemoiety, such as biotin, which can then be bound by a fourth molecule,such as enzyme-labeled streptavidin, or other proteins. (Akerstrom, JImmunol 135:2589 (1985); Chaubert, Mod Pathol 10:585 (1997)).

Throughout any particular assay, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, volume of solution, concentrations, and the like. Usually,the assays will be carried out at ambient temperature, although they canbe conducted over a range of temperatures.

Non-Competitive Binding Assays:

Binding assays can be of the non-competitive type in which the amount ofcaptured myostatin is directly measured. For example, in one preferred“sandwich” assay, the binding agent can be bound directly to a solidsubstrate where it is immobilized. These immobilized agents then bind tomyostatin present in the test sample. The immobilized myostatin is thenbound with a labeling agent, such as a labeled antibody againstmyostatin, which can be detected. In another preferred “sandwich” assay,a second agent specific for the binding agent can be added whichcontains a detectable moiety, such as biotin, to which a third labeledmolecule can specifically bind, such as streptavidin. (See, Harlow andLane, Antibodies, A Laboratory Manual, Ch 14, Cold Spring HarborLaboratory, NY (1988), which is incorporated herein by reference).

Competitive Binding Assays:

Binding assays can be of the competitive type. The amount of myostatinpresent in the sample is measured indirectly by measuring the amount ofmyostatin displaced, or competed away, from a binding agent by themyostatin present in the sample. In one preferred competitive bindingassay, a known amount of myostatin, usually labeled, is added to thesample and the sample is then contacted with the binding agent. Theamount of labeled myostatin bound to the binding agent is inverselyproportional to the concentration of myostatin present in the sample,(following the protocols found in, for example Harlow and Lane,Antibodies, A Laboratory Manual, Ch 14, pp. 579-583, supra).

In another preferred competitive binding assay, the binding agent isimmobilized on a solid substrate. The amount of myostastin bound to thebinding agent may be determined either by measuring the amount ofmyostatin present in a myostatin/binding agent complex, or alternativelyby measuring the amount of remaining uncomplexed myostatin.

Other Binding Assays

The present invention also provides Western blot methods to detect orquantify the presence of myostatin in a sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight and transferring the proteins to a suitable solidsupport, such as nitrocellulose filter, a nylon filter, or derivatizednylon filter. The sample is incubated with the binding agents orfragments thereof that bind myostatin and the resulting complex isdetected. These binding agents may be directly labeled or alternativelymay be subsequently detected using labeled antibodies that specificallybind to the binding agent.

Diagnostic Assays

The binding agents or fragments thereof of the present invention may beuseful for the diagnosis of conditions or diseases characterized byincreased amounts of myostatin. Diagnostic assays for high levels ofmyostatin include methods utilizing a binding agent and a label todetect myostatin in human body fluids, extracts of cells or specifictissue extracts. For example, serum levels of myostatin may be measuredin an individual over time to determine the onset of muscle wastingassociated with aging or inactivity, as described, for example, inYarasheski et al., supra. Increased myostatin levels were shown tocorrelate with average decreased muscle mass and fat-free mass in groupsof men and women of increasing ages (Yarasheski et al., supra). Thebinding agents of the present invention may be useful for monitoringincreases or decreases in the levels of myostatin with a givenindividual over time, for example. The binding agents can be used insuch assays with or without modification. In a preferred diagnosticassay, the binding agents will be labeled by attaching, e.g., a label ora reporter molecule. A wide variety of labels and reporter molecules areknown, some of which have been already described herein. In particular,the present invention is useful for diagnosis of human disease.

A variety of protocols for measuring myostatin proteins using bindingagents of myostatin are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescenceactivated cell sorting (FACS).

For diagnostic applications, in certain embodiments the binding agentsof the present invention typically will be labeled with a detectablemoiety. The detectable moiety can be any one that is capable ofproducing, either directly or indirectly, a detectable signal. Forexample, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, suchas alkaline phosphatase, βgalactosidase, or horseradish peroxidase(Bayer et al., Meth Enz, 184: 138 (1990)).

Pharmaceutical Compositions

Pharmaceutical compositions of myostatin binding agents such aspeptibodies described herein are within the scope of the presentinvention. Such compositions comprise a therapeutically orprophylactically effective amount of a myostatin binding agent,fragment, variant, or derivative thereof as described herein, inadmixture with a pharmaceutically acceptable agent. In a preferredembodiment, pharmaceutical compositions comprise antagonist bindingagents that inhibit myostatin partially or completely in admixture witha pharmaceutically acceptable agent. Typically, the myostatin bindingagents will be sufficiently purified for administration to an animal.

The pharmaceutical composition may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.Suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates, other organic acids); bulking agents(such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides and other carbohydrates (such as glucose, mannose, ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring; flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides(preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the binding agent.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefore. In oneembodiment of the present invention, binding agent compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the binding agent product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions may be selected for inhalation or forenteral delivery such as orally, aurally, opthalmically, rectally, orvaginally. The preparation of such pharmaceutically acceptablecompositions is within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired binding agent in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which a binding agent is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes, that providesfor the controlled or sustained release of the product which may then bedelivered via a depot injection. Hyaluronic acid may also be used, andthis may have the effect of promoting sustained duration in thecirculation. Other suitable means for the introduction of the desiredmolecule include implantable drug delivery devices.

In another aspect, pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions. In anotherembodiment, a pharmaceutical composition may be formulated forinhalation. For example, a binding agent may be formulated as a drypowder for inhalation. Polypeptide or nucleic acid molecule inhalationsolutions may also be formulated with a propellant for aerosol delivery.In yet another embodiment, solutions may be nebulized. Pulmonaryadministration is further described in PCT Application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, binding agentmolecules that are administered in this fashion can be formulated withor without those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized Additional agents can be includedto facilitate absorption of the binding agent molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

Pharmaceutical compositions for oral administration can also beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Another pharmaceutical composition may involve an effective quantity ofbinding agent in a mixture with non-toxic excipients that are suitablefor the manufacture of tablets. By dissolving the tablets in sterilewater, or other appropriate vehicle, solutions can be prepared in unitdose form. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving binding agent molecules insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. Seefor example, PCT/US93/00829 that describes controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. Additional examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983),poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res., 15:167-277, (1981); Langer et al., Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., PNAS (USA),82:3688 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the bindingagent molecule is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.In other embodiments, the dosage may range from 0.1 mg/kg up to about100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100mg/kg.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The exact dosage will be determined in light of factors related to thesubject requiring treatment. Dosage and administration are adjusted toprovide sufficient levels of the active compound or to maintain thedesired effect. Factors that may be taken into account include theseverity of the disease state, the general health of the subject, theage, weight, and gender of the subject, time and frequency ofadministration, drug combination(s), reaction sensitivities, andresponse to therapy. Long-acting pharmaceutical compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

The frequency of dosing will depend upon the pharmacokinetic parametersof the binding agent molecule in the formulation used. Typically, acomposition is administered until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as multiple doses (at the same or differentconcentrations/dosages) over time, or as a continuous infusion. Furtherrefinement of the appropriate dosage is routinely made. Appropriatedosages may be ascertained through use of appropriate dose-responsedata.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, urethral, vaginal, or rectalmeans, by sustained release systems or by implantation devices. Wheredesired, the compositions may be administered by bolus injection orcontinuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or another appropriatematerial on to which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In some cases, it may be desirable to use pharmaceutical compositions inan ex vivo manner. In such instances, cells, tissues, or organs thathave been removed from the patient are exposed to the pharmaceuticalcompositions after which the cells, tissues and/or organs aresubsequently implanted back into the patient.

In other cases, a binding agent of the present invention such as apeptibody can be delivered by implanting certain cells that have beengenetically engineered, using methods such as those described herein, toexpress and secrete the polypeptide. Such cells may be animal or humancells, and may be autologous, heterologous, or xenogeneic. Optionally,the cells may be immortalized. In order to decrease the chance of animmunological response, the cells may be encapsulated to avoidinfiltration of surrounding tissues. The encapsulation materials aretypically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Pharmaceutical compositions containing the binding agents of the presentinvention are administered to a subject to treat any myostatin-relateddisorders. These include muscle-wasting disorders including but notlimited to muscular dystrophy, muscle wasting in cancer, AIDS, muscleatrophy, rheumatoid arthritis, renal failure/uremia, chronic heartfailure, prolonged bed-rest, spinal chord injury, stroke, and agingrelated sarcopenia. In addition these compositions are administed totreat obesity, diabetes, hyperglycemia, and increase bone density,

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

Example 1 Identification of Myostatin-Binding Peptides

Three filamentous phage libraries, TN8-IX (5×10⁹ independenttransformants), TN12-I (1.4×10⁹ independent transformants), and linear(2.3×10⁹ independent transformants) (Dyax Corp.) were used to select formyostatin binding phage. Each library was incubated on myostatin-coatedsurfaces and subjected to different panning conditions: non-specificelution, and specific elution using recombinant human activin receptorIIB/Fc chimera (R&D Systems, Inc., Minneapolis, Minn.), or myostatinpropeptide elution as described below. For all three libraries, thephages were eluted in a non-specific manner for the first round ofselection, while the receptor and promyostatin was used in the secondand third rounds of selection. The selection procedures were carried outas described below.

Preparation of Myostatin

Myostatin protein was produced recombinantly in the E. coli K-12 strain2596 (ATCC #202174) as follows. Polynucleotides encoding the humanpromyostatin molecule were cloned into the pAMG21 expression vector(ATCC No. 98113), which was derived from expression vector pCFM1656(ATCC No. 69576) and the expression vector system described in U.S. Pat.No. 4,710,473, by following the procedure described in publishedInternational Patent Application WO 00/24782. The polynucleotidesencoding promyostatin were obtained from a mammalian expression vector.The coding region was amplified using a standard PCR method and thefollowing PCR primers to introduce the restriction site for NdeI andBamHI.

5′primer: (Seq ID No: 292) 5′-GAGAGAGAGCATATGAATGAGAACAGTGAGCAAAAAG-3′3′primer: (Seq ID No: 293) 5′-AGAGAGGGATCCATTATGAGCACCCACAGCGGTC-3′

The PCR product and vector were digested with both enzymes, mixed andligated. The product of the ligation was transformed into E. coli strain#2596. Single colonies were checked microscopically for recombinantprotein expression in the form of inclusion bodies. The plasmid wasisolated and sequenced through the coding region of the recombinant geneto verify genetic fidelity.

Bacterial paste was generated from a 10L fermentation using a batchmethod at 37° C. The culture was induced with HSL at a cell density of9.6 OD₆₀₀ and harvested six hours later at a density of 104 OD₆₀₀. Thepaste was stored at −80° C. E. coli paste expressing promyostatin waslysed in a microfluidizer at 16,000 psi, centrifuged to isolate theinsoluble inclusion body fraction. Inclusion bodies were resuspended inguanidine hydrochloride containing dithiothreitol and solubilized atroom temperature. This was then diluted 30 fold in an aqueous buffer.The refolded promyostatin was then concentrated and buffer exchangedinto 20 mM Tris pH 8.0, and applied to an anion exchange column. Theanion exchange column was eluted with an increasing sodium chloridegradient. The fractions containing promyostatin were pooled. Thepromyostatin produced in E. coli is missing the first 23 amino acids andbegins with a methionine before the residue 24 asparagine. To producemature myostatin, the pooled promyostatin was enzymatically cleavedbetween the propeptide and mature myostatin C terminal. The resultingmixture was then applied to a C4-rpHPLC column using a increasinggradient of acetonitrile containing 0.1% trifluoroacetic acid. Fractionscontaining mature myostatin were pooled and dried in a speed-vac.

The recombinant mature myostatin produced from E. coli was tested in themyoblast C2C12 based assay described below and found to be fully activewhen compared with recombinant murine myostatin commercially produced ina mammalian cell system (R&D Systems, Inc., Minneapolis, Minn.). The E.coli-produced mature myostatin was used in the phage-display andscreening assays described below.

Preparation of Myostatin-Coated Tubes

Myostatin was immobilized on 5 ml Immuno™ Tubes (NUNC) at aconcentration of 8 ug of myostatin protein in 1 ml of 0.1M sodiumcarbonate buffer (pH 9.6). The myostatin-coated Immuno™ Tube wasincubated with orbital shaking for 1 hour at room temperature.Myostatin-coated Immuno™ Tube was then blocked by adding 5 ml of 2%milk-PBS and incubating at room temperature for 1 hour with rotation.The resulting myostatin-coated Immuno™ Tube was then washed three timeswith PBS before being subjected to the selection procedures. AdditionalImmuno™ Tubes were also prepared for negative selections (no myostatin).For each panning condition, five to ten Immuno™ Tubes were subjected tothe above procedure except that the Immuno™ Tubes were coated with 1 mlof 2% BSA-PBS instead of myostatin protein.

Negative Selection

For each panning condition, about 100 random library equivalents forTN8-IX and TN12-I libraries (5×10¹¹ pfu for TN8-IX, and 1.4×10¹¹ pfu forTN12-I) and about 10 random library equivalents for the linear library(2.3×10¹⁰ pfu) were aliquoted from the library stock and diluted to 1 mlwith PBST (PBS with 0.05% Tween-20). The 1 ml of diluted library stockwas added to an Immuno™ Tube prepared for the negative selection, andincubated for 10 minutes at room temperature with orbital shaking. Thephage supernatant was drawn out and added to the second Immuno™ Tube foranother negative selection step. In this way, five to ten negativeselection steps were performed.

Selection for Myostatin Binding

After the last negative selection step above, the phage supernatant wasadded to the prepared myostatin coated Immuno™ Tubes. The Immuno™ Tubewas incubated with orbital shaking for one hour at room temperature,allowing specific phage to bind to myostatin. After the supernatant wasdiscarded, the Immuno™ Tube was washed about 15 times with 2% milk-PBS,10 times with PBST and twice with PBS for the three rounds of selectionwith all three libraries (TN8-IX, TN12-I, and Linear libraries) exceptthat for the second round of selections with TN8-IX and TN12-Ilibraries, the Immuno™ Tube was washed about 14 times with 2% milk-PBS,twice with 2% BSA-PBS, 10 times with PBST and once with PBS.

Non-Specific Elution

After the last washing step, the bound phages were eluted from theImmuno™ Tube by adding 1 ml of 100 mM triethylamine solution (Sigma, St.Louis, Mo.) with 10-minute incubation with orbital shaking. The pH ofthe phage containing solution was then neutralized with 0.5 ml of 1 MTris-HCl (pH 7.5).

Receptor (Human Activin Receptor) Elution of Bound Phage

For round 2 and 3, after the last washing step, the bound phages wereeluted from the Immuno™ Tube by adding 1 ml of 1 μM of receptor protein(recombinant human activin receptor IIB/Fc chimera, R&D Systems, Inc.,Minneapolis, Minn.) with a 1-hour incubation for each condition.

Propeptide Elution of Bound Phage

For round 2 and 3, after the last washing step, the bound phages wereeluted from the Immuno™ Tube by adding 1 ml of 1 μM propeptide protein(made as described above) with a 1-hour incubation for each condition.

Phage Amplification

Fresh E. coli. (XL-1 Blue MRF′) culture was grown to OD₆₀₀=0.5 in LBmedia containing 12.5 ug/ml tetracycline. For each panning condition, 20ml of this culture was chilled on ice and centrifuged. The bacteriapellet was resuspended in 1 ml of the min A salts solution.

Each mixture from different elution methods was added to a concentratedbacteria sample and incubated at 37° C. for 15 minutes. 2 ml of NZCYMmedia (2× NZCYM, 50 ug/ml Ampicillin) was added to each mixture andincubated at 37° C. for 15 minutes. The resulting 4 ml solution wasplated on a large NZCYM agar plate containing 50 ug/ml ampicillin andincubated overnight at 37° C.

Each of the bacteria/phage mixture that was grown overnight on a largeNZCYM agar plate was scraped off in 35 ml of LB media, and the agarplate was further rinsed with additional 35 ml of LB media. Theresulting bacteria/phage mixture in LB media was centrifuged to pelletthe bacteria away. 50 ul of the phage supernatant was transferred to afresh tube, and 12.5 ml of PEG solution (20% PEG8000, 3.5M ammoniumacetate) was added and incubated on ice for 2 hours to precipitatephages. The precipitated phages were centrifuged down and resuspended in6 ml of the phage resuspension buffer (250 mM NaCl, 100 mM Tris pH8, 1mM EDTA). This phage solution was further purified by centrifuging awaythe remaining bacteria and precipitating the phage for the second timeby adding 1.5 ml of the PEG solution. After a centrifugation step, thephage pellet was resuspended in 400 ul of PBS. This solution wassubjected to a final centrifugation to rid of remaining bacteria debris.The resulting phage preparation was titered by a standard plaqueformation assay (Molecular Cloning, Maniatis et al., 3^(rd) Edition).

Additional Rounds of Selection and Amplification

In the second round, the amplified phage (10¹¹ pfu) from the first roundwas used as the input phage to perform the selection and amplificationsteps. The amplified phage (10¹¹ pfu) from the second round in turn wasused as the input phage to perform third round of selection andamplification. After the elution steps of the third round, a smallfraction of the eluted phage was plated out as in the plaque formationassay above. Individual plaques were picked and placed into 96 wellmicrotiter plates containing 100 ul of TE buffer in each well. Thesemaster plates were incubated at 4° C. overnight to allow phages to eluteinto the TE buffer.

Clonal Analysis

Phage ELISA

The phage clones were subjected to phage ELISA and then sequenced. Thesequences were ranked as discussed below.

Phage ELISA was performed as follows. An E. Coli XL-1 Blue MRF′ culturewas grown until OD₆₀₀ reached 0.5. 30 ul of this culture was aliquotedinto each well of a 96 well microtiter plate. 10 ul of eluted phage wasadded to each well and allowed to infect bacteria for 15 min at roomtemperature. About 120 ul of LB media containing 12.5 ug/ml oftetracycline and 50 ug/ml of ampicillin were added to each well. Themicrotiter plate was then incubated with shaking overnight at 37° C.Myostatin protein (2 ug/ml in 0.1M sodium carbonate buffer, pH 9.6) wasallowed to coat onto a 96 well Maxisorp™ plates (NUNC) overnight at 4°C. As a control, a separate Maxisorp™ plate was coated with 2% BSAprepared in PBS.

On the following day, liquid in the protein coated Maxisorp™ plates wasdiscarded, washed three times with PBS and each well was blocked with300 ul of 2% milk solution at room temperature for 1 hour. The milksolution was discarded, and the wells were washed three times with thePBS solution. After the last washing step, about 50 ul of PBST-4% milkwas added to each well of the protein-coated Maxisorp™ plates. About 50ul of overnight cultures from each well in the 96 well microtiter platewas transferred to the corresponding wells of the myostatin coatedplates as well as the control 2% BSA coated plates. The 100 ul mixturein the two kinds of plates were incubated for 1 hour at roomtemperature. The liquid was discarded from the Maxisorp™ plates, and thewells were washed about three times with PBST followed by two times withPBS. The HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech)was diluted to about 1:7,500, and 100 ul □of the diluted solution wasadded to each well of the Maxisorp™ plates for 1 hour incubation at roomtemperature. The liquid was again discarded and the wells were washedabout three times with PBST followed by two time with PBS. 100 ul ofLumiGlo™ Chemiluminescent substrate (KPL) was added to each well of theMaxisorp™ plates and incubated for about 5 minutes for reaction tooccur. The chemiluminescent unit of the Maxisorp™ plates was read on aplate reader (Lab System).

Sequencing of the Phage Clones

For each phage clone, the sequencing template was prepared by a PCRmethod. The following oligonucleotide pair was used to amplify a 500nucleotide fragment: primer #1: 5′-CGGCGCAACTATCGGTATCAAGCTG-3′ (Seq IDNo: 294) and primer #2: 5′-CATGTACCGTAACACTGAGTTTCGTC-3′(Seq ID No:295). The following mixture was prepared for each clone.

Reagents Volume (μL)/tube distilled H₂O 26.25 50% glycerol 10 10X PCRBuffer (w/o MgCl₂) 5 25 mM MgCl₂ 4 10 mM dNTP mix 1 100 μM primer 1 0.25100 μM primer 2 0.25 Taq polymerase 0.25 Phage in TE (section 4) 3 Finalreaction volume 50

A thermocycler (GeneAmp PCR System 9700, Applied Biosystem) was used torun the following program: [94° C. for 5 min; 94° C. for 30 sec, 55° C.for 30 sec, 72° C. for 45 sec.]×30 cycles; 72° C. for 7 min; cool to 4°C. The PCR product from each reaction was cleaned up using the QIAquickMultiwell PCR Purification kit (Qiagen), following the manufacturer'sprotocol. The PCR cleaned up product was checked by running 10 ul ofeach PCR reaction mixed with 1 ul of dye (10×BBXS agarose gel loadingdye) on a 1% agarose gel. The remaining product was then sequenced usingthe ABI 377 Sequencer (Perkin Elmer) following the manufacturerrecommended protocol.

Sequence Ranking and Analysis

The peptide sequences that were translated from the nucleotide sequenceswere correlated to ELISA data. The clones that showed highchemiluminescent units in the myostatin-coated wells and lowchemiluminescent units in the 2% BSA-coated wells were identified. Thesequences that occurred multiple times were identified. Candidatesequences chosen based on these criteria were subjected to furtheranalysis as peptibodies. Approximately 1200 individual clones wereanalyzed. Of these approximately 132 peptides were chosen for generatingthe peptibodies of the present invention. These are shown in Table Tbelow. The peptides having SEQ ID NO: 1 to 129 were used to generatepeptibodies of the same name. The peptides having SEQ ID NO: 130 to 141shown in Table 1 comprise two or more peptides from SEQ ID NO: 1 to 132attached by a linker sequence. SEQ ID NO: 130 to 141 were also used togenerate peptibodies of the same name.

Consensus sequences were determined for the TN-8 derived group ofpeptides. These are as follows:

KDXCXXWHWMCKPX (Seq ID No: 142) WXXCXXXGFWCXNX (Seq ID No: 143)IXGCXWWDXXCYXX (Seq ID No: 144) XXWCVSPXWFCXXX (Seq ID No: 145)XXXCPWFAXXCVDW (Seq ID No: 146)For all of the above consensus sequences, the underlined “coresequences” from each consensus sequence are the amino acid which alwaysoccur at that position. “X” refers to any naturally occurring ormodified amino acid. The two cysteines contained with the core sequenceswere fixed amino acids in the TN8-TX library.

TABLE I SEQ. PEPTIBODY ID PEPTIDE NAME No SEQUENCE Myostatin-TN8-Con1  1 KDKCKMWHWMCKPP Myostatin-TN8-Con2   2 KDLCAMWHWMCKPPMyostatin-TN8-Con3   3 KDLCKMWKWMCKPP Myostatin-TN8-Con4   4KDLCKMWHWMCKPK Myostatin-TN8-Con5   5 WYPCYEFHFWCYDL Myostatin-TN8-Con6  6 WYPCYEGHFWCYDL Myostatin-TN8-Con7   7 IFGCKWWDVQCYQFMyostatin-TN8-Con8   8 IFGCKWWDVDCYQF Myostatin-TN8-Con9   9ADWCVSPNWFCMVM Myostatin-TN8-Con10  10 HKFCPWWALFCWDF Myostatin-TN8-1 11 KDLCKMWHWMCKPP Myostatin-TN8-2  12 IDKCAIWGWMCPPL Myostatin-TN8-3 13 WYPCGEFGMWCLNV Myostatin-TN8-4  14 WFTCLWNCDNE Myostatin-TN8-5  15HTPCPWFAPLCVEW Myostatin-TN8-6  16 KEWCWRWKWMCKPE Myostatin-TN8-7  17FETCPSWAYFCLDI Myostatin-TN8-8  18 AYKCEANDWGCWWL Myostatin-TN8-9  19NSWCEDQWHRCWWL Myostatin-TN8-10  20 WSACYAGHFWCYDL Myostatin-TN8-11  21ANWCVSPNWFCMVM Myostatin-TN8-12  22 WTECYQQEFWCWNL Myostatin-TN8-13  23ENTCERWKWMCPPK Myostatin-TN8-14  24 WLPCHQEGFWCMNF Myostatin-TN8-15  25STMCSQWHWMCNPF Myostatin-TN8-16  26 IFGCHWWDVDCYQF Myostatin-TN8-17  27IYGCKWWDIQCYDI Myostatin-TN8-18  28 PDWCIDPDWWCKFW Myostatin-TN8-19  29QGHCTRWPWMCPPY Myostatin-TN8-20  30 WQECYREGFWCLQT Myostatin-TN8-21  31WFDCYGPGFKCWSP Myostatin-TN8-22  32 GVRCPKGHLWCLYP Myostatin-TN8-23  33HWACGYWPWSCKWV Myostatin-TN8-24  34 GPACHSPWWWCVFG Myostatin-TN8-25  35TTWCISPMWFCSQQ Myostatin-TN8-26  36 HKFCPPWAIFCWDF Myostatin-TN8-27  37PDWCVSPRWYCNMW Myostatin-TN8-28  38 VWKCHWFGMDCEPT Myostatin-TN8-29  39KKHCQTWTWMCAPK Myostatin-TN8-30  40 WFQCGSTLFWCYNL Myostatin-TN8-31  41WSPCYDHYFYCYTI Myostatin-TN8-32  42 SWMCGFFKEVCMWV Myostatin-TN8-33  43EMLCMIHPVFCNPH Myostatin-TN8-34  44 LKTCNLWPWMCPPL Myostatin-TN8-35  45VVGCKWYEAWCYNK Myostatin-TN8-36  46 PIHCTQWAWMCPPT Myostatin-TN8-37  47DSNCPWYFLSCVIF Myostatin-TN8-38  48 HIWCNLAMMKCVEM Myostatin-TN8-39  49NLQCIYFLGKCIYF Myostatin-TN8-40  50 AWRCMWFSDVCTPG Myostatin-TN8-41  51WFRCFLDADWCTSV Myostatin-TN8-42  52 EKICQMWSWMCAPP Myostatin-TN8-43  53WFYCHLNKSECTEP Myostatin-TN8-44  54 FWRCAIGIDKCKRV Myostatin-TN8-45  55NLGCKWYEVWCFTY Myostatin-TN8-46  56 IDLCNMWDGMCYPP Myostatin-TN8-47  57EMPCNIWGWMCPPV Myostatin-TN12-1  58 WFRCVLTGIVDWSECFGL Myostatin-TN12-2 59 GFSCTFGLDEFYVDCSPF Myostatin-TN12-3  60 LPWCHDQVNADWGFCMLWMyostatin-TN12-4  61 YPTCSEKFWIYGQTCVLW Myostatin-TN12-5  62LGPCPIHHGPWPQYCVYW Myostatin-TN12-6  63 PFPCETHQISWLGHCLSFMyostatin-TN12-7  64 HWGCEDLMWSWHPLCRRP Myostatin-TN12-8  65LPLCDADMMPTIGFCVAY Myostatin-TN12-9  66 SHWCETTFWMNYAKCVHAMyostatin-TN12-10  67 LPKCTHVPFDQGGFCLWY Myostatin-TN12-11  68FSSCWSPVSRQDMFCVFY Myostatin-TN12-13  69 SHKCEYSGWLQPLCYRPMyostatin-TN12-14  70 PWWCQDNYVQHMLHCDSP Myostatin-TN12-15  71WFRCMLMNSFDAFQCVSY Myostatin-TN12-16  72 PDACRDQPWYMFMGCMLGMyostatin-TN12-17  73 FLACFVEFELCFDS Myostatin-TN12-18  74SAYCIITESDPYVLCVPL Myostatin-TN12-19  75 PSICESYSTMWLPMCQHNMyostatin-TN12-20  76 WLDCHDDSWAWTKMCRSH Myostatin-TN12-21  77YLNCVMMNTSPFVECVFN Myostatin-TN12-22  78 YPWCDGFMIQQGITCMFYMyostatin-TN12-23  79 FDYCTWLNGFKDWKCWSR Myostatin-TN12-24  80LPLCNLKEISHVQACVLF Myostatin-TN12-25  81 SPECAFARWLGIEQCQRDMyostatin-TN12-26  82 YPQCFNLHLLEWTECDWF Myostatin-TN12-27  83RWRCEIYDSEFLPKCWFF Myostatin-TN12-28  84 LVGCDNVWHRCKLFMyostatin-TN12-29  85 AGWCHVWGEMFGMGCSAL Myostatin-TN12-30  86HHECEWMARWMSLDCVGL Myostatin-TN12-31  87 FPMCGIAGMKDFDFCVWYMyostatin-TN12-32  88 RDDCTFWPEWLWKLCERP Myostatin-TN12-33  89YNFCSYLFGVSKEACQLP Myostatin-TN12-34  90 AHWCEQGPWRYGNICMAYMyostatin-TN12-35  91 NLVCGKTSAWGDEACARA Myostatin-TN12-36  92HNVCTIMGPSMKWFCWND Myostatin-TN12-37  93 NDLCAMWGWRNTIWCQNSMyostatin-TN12-38  94 PPFCQNDNDMLQSLCKLL Myostatin-TN12-39  95WYDCNVPNELLSGLCRLF Myostatin-TN12-40  96 YGDCDQNHWMWPFTCLSLMyostatin-TN12-41  97 GWMCHFDLHDWGATCQPD Myostatin-TN12-42  98YFHCMFGGHEFEVHCESF Myostatin-TN12-43  99 AYWCWHGQCVRF Myostatin-Linear-1100 SEHWTFTDWDGNEWWVRPF Myostatin-Linear-2 101 MEMLDSLFELLKDMVPISKAMyostatin-Linear-3 102 SPPEEALMEWLGWQYGKFT Myostatin-Linear-4 103SPENLLNDLYILMTKQEWYG Myostatin-Linear-5 104 FHWEEGIPFHVVTPYSYDRMMyostatin-Linear-6 105 KRLLEQFMNDLAELVSGHS Myostatin-Linear-7 106DTRDALFQEFYEFVRSRLVI Myostatin-Linear-8 107 RMSAAPRPLTYRDIMDQYWHMyostatin-Linear-9 108 NDKAHFFEMFMFDVHNFVES Myostatin-Linear-10 109QTQAQKIDGLWELLQSIRNQ Myostatin-Linear-11 110 MLSEFEEFLGNLVHRQEAMyostatin-Linear-12 111 YTPKMGSEWTSFWHNRIHYL Myostatin-Linear-13 112LNDTLLRELKMVLNSLSDMK Myostatin-Linear-14 113 FDVERDLMRWLEGFMQSAATMyostatin-Linear-15 114 HHGWNYLRKGSAPQWFEAWV Myostatin-Linear-16 115VESLHQLQMWLDQKLASGPH Myostatin-Linear-17 116 RATLLKDFWQLVEGYGDNMyostatin-Linear-18 117 EELLREFYRFVSAFDY Myostatin-Linear-19 118GLLDEFSHFIAEQFYQMPGG Myostatin-Linear-20 119 YREMSMLEGLLDVLERLQHYMyostatin-Linear-21 120 HNSSQMLLSELIMLVGSMMQ Myostatin-Linear-22 121WREHFLNSDYIRDKLIAIDG Myostatin-Linear-23 122 QFPFYVFDDLPAQLEYWIAMyostatin-Linear-24 123 EFFHWLHNHRSEVNHWLDMN Myostatin-Linear-25 124EALFQNFFRDVLTLSEREY Myostatin-Linear-26 125 QYWEQQWMTYFRENGLHVQYMyostatin-Linear-27 126 NQRMMLEDLWRIMTPMFGRS Myostatin-Linear-29 127FLDELKAELSRHYALDDLDE Myostatin-Linear-30 128 GKLIEGLLNELMQLETFMPDMyostatin-Linear-31 129 ILLLDEYKKDWKSWF Myostatin-2xTN8- 130QGHCTRWPWMCPPYGSGSATGGS 19 kc GSTASSGSGSATGQGHCTRWPWM CPPYMyostatin-2xTN8- 131 WYPCYEGHFWCYDLGSGSTASSG con6 SGSATGWYPCYEGHFWCYDLMyostatin-2xTN8- 132 HTPCPWFAPLCVEWGSGSATGGSG 5 kcSTASSGSGSATGHTPCPWFAPLCV EW Myostatin-2xTN8- 133 PDWCIDPDWWCKFWGSGSATGGS18 kc GSTASSGSGSATGPDWCIDPDWW CKFW Myostatin-2xTN8- 134ANWCVSPNWFCMVMGSGSATGG 11 kc SGSTASSGSGSATGANWCVSPNWF CMVMMyostatin-2xTN8- 135 PDWCIDPDWWCKFWGSGSATGGS 25 kcGSTASSGSGSATGPDWCIDPDWW CKFW Myostatin-2xTN8- 136 HWACGYWPWSCKWVGSGSATGG23 kc SGSTASSGSGSATGHWACGYWPW SCKWV Myostatin-TN8- 137KKHCQIWTWMCAPKGSGSATGGS 29-19 kc GSTASSGSGSATGQGHCTRWPWM CPPYMyostatin-TN8- 138 QGHCTRWPWMCPPYGSGSATGGS 19-29 kcGSTASSGSGSATGKKHCQIWTWM CAPK Myostatin-TN8- 139 KKHCQIWTWMCAPKGSGSATGGS29-19 kn GSTASSGSGSATGQGHCTRWPWM CPPY Myostatin-TN8- 140KKHCQIWTWMCAPKGGGGGGGG 29-19-8g QGHCTRWPWMCPPY Myostatin-TN8- 141QGHCTRWPWMCPPYGGGGGGKK 19-29-6gc HCQIWTWMCAPK

Example 2 Generating Peptibodies

Construction of DNA Encoding Peptide-Fc Fusion Proteins

Peptides capable of binding myostatin were used alone or in combinationwith each other to construct fusion proteins in which a peptide wasfused to the Fc domain of human IgG1. The amino acid sequence of the Fcportion of each peptibody is as follows (from amino terminus to carboxylterminus):

(Seq ID No: 296) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

The peptide was fused in the N configuration (peptide was attached tothe N-terminus of the Fc region), the C configuration (peptide wasattached to the C-terminus of the Fc region), or the N,C configuration(peptide attached both at the N and C terminus of the Fc region).Separate vectors were used to express N-terminal fusions and C-terminalfusions. Each peptibody was constructed by annealing pairs ofoligonucleotides (“oligos”) to the selected phage nucleic acid togenerate a double stranded nucleotide sequence encoding the peptide.These polynucleotide molecules were constructed as ApaL to XhoIfragments. The fragments were ligated into either the pAMG21-FcN-terminal vector for the N-terminal orientation, or thepAMG21-Fc-C-terminal vector for the C-terminal orientation which hadbeen previously digested with ApaLI and XhoI. The resulting ligationmixtures were transformed by electroporation into E. coli strain 2596 or4167 cells (a hsdR- variant of strain 2596 cells) using standardprocedures. Clones were screened for the ability to produce therecombinant protein product and to possess the gene fusion having acorrect nucleotide sequence. A single such clone was selected for eachof the modified peptides.

Many of constructs were created using an alternative vector designatedpAMG21-2xBs-N(ZeoR) Fc. This vector is similar to the above-describedvector except that the vector digestion was performed with BsmBI. Someconstructs fused peptide sequences at both ends of the Fc. In thosecases the vector was a composite of pAMG21-2xBs-N(ZeoR) Fc andpAMG21-2xBs-C-Fc.

Construction of pAMG21

Expression plasmid pAMG21 (ATCC No. 98113) is derived from expressionvector pCFM1656 (ATCC No. 69576) and the expression vector systemdescribed in U.S. Pat. No. 4,710,473, by following the proceduredescribed in published International Patent Application WO 00/24782, allof which are incorporated herein by reference.

Fc N-terminal Vector

The Fc N-terminal vector was constructed using the pAMG21 Fc_Gly5_Tpovector as a template. A 5′ PCR primer (below) was designed to remove theTpo peptide sequence in pAMG Tpo Gly5 and replace it with a polylinkercontaining ApaLI and XhoI sites. Using this vector as a template, PCRwas performed with Expand Long Polymerase, using the following 5′ primerand a universal 3′ primer:

5′ primer: (Seq ID No: 297)5′-ACAAACAAACATATGGGTGCACAGAAAGCGGCCGCAAAAAAACTCGAGGGTGGAGGCGGTGGGGACA-3′ 3′ primer: (Seq ID No: 298)5′-GGTCATTACTGGACCGGATC-3′

The resulting PCR product was gel purified and digested with restrictionenzymes NdeI and BsrGI. Both the plasmid and the polynucleotide encodingthe peptide of interest together with its linker were gel purified usingQiagen (Chatsworth, Calif.) gel purification spin columns. The plasmidand insert were then ligated using standard ligation procedures, and theresulting ligation mixture was transformed into E. coli cells (strain2596). Single clones were selected and DNA sequencing was performed. Acorrect clone was identified and this was used as a vector source forthe modified peptides described herein.

Construction of Fc C-Terminal Vector

The Fc C-terminal vector was constructed using pAMG21 Fc_Gly5_Tpo vectoras a template. A 3′ PCR primer was designed to remove the Tpo peptidesequence and to replace it with a polylinker containing ApaLI and XhoIsites. PCR was performed with Expand Long Polymerase using a universal5′ primer and the 3′ primer.

5′ Primer: (Seq ID No: 299) 5′-CGTACAGGTTTACGCAAGAAAATGG-3′ 3′ Primer:(Seq ID No: 300) 5′-TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGCCGCTTTCTGTGCACCACCACCTCCACCTTTAC-3′

The resulting PCR product was gel purified and digested with restrictionenzymes BsrGI and BamHI. Both the plasmid and the polynucleotideencoding each peptides of interest with its linker were gel purified viaQiagen gel purification spin columns. The plasmid and insert were thenligated using standard ligation procedures, and the resulting ligationmixture was transformed into E. coli (strain 2596) cells. Strain 2596(ATCC #202174) is a strain of E. coli K-12 modified to contain the luxpromoter and two lambda temperature sensitive repressors, the cI857s7and the lac I^(Q) repressor. Single clones were selected and DNAsequencing was performed. A correct clone was identified and used as asource of each peptibody described herein.

Expression in E. coli.

Cultures of each of the pAMG21-Fc fusion constructs in E. coli strain2596 were grown at 37° C. in Terrific Broth medium (See Tartof andHobbs, “Improved media for growing plasmid and cosmid clones”, BethesdaResearch Labs Focus, Volume 9, page 12, 1987, cited in aforementionedSambrook et al. reference). Induction of gene product expression fromthe luxPR promoter was achieved following the addition of the syntheticautoinducer, N-(3-oxohexanoyl)-DL-homoserine lactone, to the culturemedium to a final concentration of 20 nanograms per milliliter (ng/ml).Cultures were incubated at 37° C. for an additional six hours. Thebacterial cultures were then examined by microscopy for the presence ofinclusion bodies and collected by centrifugation. Refractile inclusionbodies were observed in induced cultures, indicating that the Fc-fusionswere most likely produced in the insoluble fraction in E. coli. Cellpellets were lysed directly by resuspension in Laemmli sample buffercontaining 10% β-mercaptoethanol and then analyzed by SDS-PAGE. In mostcases, an intense coomassie-stained band of the appropriate molecularweight was observed on an SDS-PAGE gel.

Folding and Purifying Peptibodies

Cells were broken in water (1/10 volume per volume) by high pressurehomogenization (3 passes at 15,000 PST) and inclusion bodies wereharvested by centrifugation (4000 RPM in 5-6B for 30 minutes). Inclusionbodies were solubilized in 6 M guanidine, 50 mM Tris, 8 mM DTT, pH 8.0for 1 hour at a 1/10 ratio at ambient temperature. The solubilizedmixture was diluted 25 times into 4 M urea, 20% glycerol, 50 mM Tris,160 mM arginine, 3 mM cysteine, 1 mM cystamine, pH 8.5. The mixture wasincubated overnight in the cold. The mixture was then dialyzed against10 mM Tris pH 8.5, 50 mM NaCl, 1.5 M urea. After an overnight dialysisthe pH of the dialysate was adjusted to pH 5 with acetic acid. Theprecipitate was removed by centrifugation and the supernatant was loadedonto a SP-Sepharose Fast Flow column equilibrated in 10 mM NaAc, 50 mMNaCl, pH 5, 4° C.). After loading the column was washed to baseline with10 mM NaAc, 50 mM NaCl, pH 5.2. The column was developed with a 20column volume gradient from 50 mM-500 mM NaCl in the acetate buffer.Alternatively, after the wash to baseline, the column was washed with 5column volumes of 10 mM sodium phosphate pH 7.0 and the column developedwith a 15 column volume gradient from 0-400 mM NaCl in phosphate buffer.Column fractions were analyzed by SDS-PAGE. Fractions containing dimericpeptibody were pooled. Fractions were also analyzed by gel filtration todetermine if any aggregate was present.

A number of peptibodies were prepared from the peptides of Table I. Thepeptides were attached to the human IgG1 Fc molecule to form thepeptibodies in Table II. Regarding the peptibodies in Table II, the Cconfiguration indicates that the peptide named was attached at theC-termini of the Fc. The N configuration indicates that the peptidenamed was attached at the N-termini of the Fc. The N,C configurationindicates that one peptide was attached at the N-termini and one at theC-termini of each Fc molecule. The 2× designation indicates that the twopeptides named were attached in tandem to each other and also attachedat the N or the C termini, or both the N,C of the Fc, separated by thelinker indicated. Two peptides attached in tandem separated by a linker,are indicated, for example, as Myostatin-TN8-29-19-8g, which indicatesthat TN8-29 peptide is attached via a (gly)₅ linker to TN8-19 peptide.The peptide(s) were attached to the Fc via a (gly)₅ linker sequenceunless otherwise specified. In some instances the peptide(s) wereattached via a k linker. The linker designated k or 1k refers to thegsgsatggsgstassgsgsatg (Seq ID No: 301) linker sequence, with kcreferring to the linker attached to the C-terminus of the Fc, and knreferring to the linker attached to the N-terminus of the Fc. In TableII below, column 4 refers to the linker sequence connecting the Fc tothe first peptide and the fifth column refers to the configuration N orC or both.

Since the Fc molecule dimerizes in solution, a peptibody constructed soas to have one peptide will actually be a dimer with two copies of thepeptide and two Fc molecules, and the 2× version having two peptides intandem will actually be a dimer with four copies of the peptide and twoFc molecules.

Since the peptibodies given in Table II are expressed in E. coli, thefirst amino acid residue is Met (M). Therefore, the peptibodies in the Nconfiguration are Met-peptide-linker-Fc, orMet-peptide-linker-peptide-linker-Fc, for example. Peptibodies in the Cconfiguration are arranged as Met-Fc-linker-peptide orMet-Fc-linker-peptide-linker-peptide, for example. Peptibodies in theC,N configuration are a combination of both, for example,Met-peptide-linker-Fe-linker-peptide.

Nucleotide sequences encoding exemplary peptibodies are provided belowin Table II. The polynucleotide sequences encoding an exemplarypeptibody of the present invention includes a nucleotide sequenceencoding the Fc polypeptide sequence such as the following:

(Seq ID No: 301) 5′-GACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA-3′

In addition, the polynucleotides encoding the (Gly)₅ linker such as thefollowing are included:

5′-GGTGGAGGTGGTGGT-3′. (Seq ID No: 302)

The polynucleotide encoding the peptibody also includes the codonencoding the methionine ATG and a stop codon such as TAA.

Therefore, the structure of the first peptibody in Table II is TN8-Con1with a C configuration and a (gly)₅ linker is as follows:M-Fc-GGGGG-KDKCKMWHWMCKPP (Seq ID No: 303). Exemplary polynucleotidesencoding this peptibody would be:

(Seq ID No: 304) 5′-ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTAAGACAAATGCAAAATGTGGCACTGGATGTGCAAACCGCCG- 3′

TABLE II Nucleotide Sequence Peptibody Name Peptide (SEQ ID NO) LMyostatin-TN8-con1 KDKCKMWHWMCKPP AAAGACAAATGCAAAATGTGGCACTG 5 gly C(SEQ ID NO: 1) GATGTGCAAACCGCCG (SEQ. ID NO: 147) Myostatin-TN8-con2KDLCAMWIIWMCKPP AAAGACCTGTGCGCTATGTGGCACTG 5 gly C (SEQ ID NO: 2)GATGTGCAAACCGCCG (SEQ. ID NO: 148) Myostatin-TN8-con3 KDLCKMWKWMCKPPAAAGACCTGTGCAAAATGTGGAAATG 5 gly C (SEQ ID NO: 3) GATGTGCAAACCGCCG(SEQ ID NO: 149) Myostatin-TN8-con4 KDLCKMWHWMCKPKAAAGACCTGTGCAAAATGTGGCACTG 5 gly C (SEQ ID NO: 4) GATGTGCAAACCGAAA(SEQ ID NO: 150) Myostatin-TN8-con5 WYPCYEFHFWCYDLTGGTACCCGTGCTACGAATTCCACTTC 5 gly C (SEQ ID NO: 5) TGGTGCTACGACCTG(SEQ ID NO: 151) Myostatin-TN8-con5 WYPCYEFHFWCYDLTGGTACCCGTGCTACGAATTCCACTTC 5 gly N (SEQ ID NO: 5) TGGTGCTACGACCTG(SEQ ID NO: 152) Myostatin-TN8-con6 WYPCYEGHFWCYDLTGGTACCCGTGCTACGAAGGTCACTT 5 gly C (SEQ ID NO: 6) CTGGTGCTACGACCTG(SEQ ID NO: 153) Myostatin-TN8-con6 WYPCYEGHFWCYDLTGGTACCCGTGCTACGAAGGTCACTT 5 gly N (SEQ ID NO: 6) CTGGTGCTACGACCTG(SEQ ID NO: 154) Myostatin-TN8-con7 IFGCKWWDVQCYQFATCTTCGGTTGCAAATGGTGGGACGT 5 gly C (SEQ ID NO: 7) TCAGTGCTACCAGTTC(SEQ. ID NO: 155) Myostatin-TN8-con8 IFGCKWWDVDCYQFATCTTCGGTTGCAAATGGTGGGACGT 5 gly C (SEQ ID NO: 8) TGACTGCTACCAGTTC(SEQ ID NO: 156) Myostatin-TN8-con8 IFGCKWWDVDCYQFATCTTCGGTTGCAAATGGTGGGACGT 5 gly N (SEQ ID NO: 8) TGACTGCTACCAGTTC(SEQ ID NO: 157) Myostatin-TN8-con9 ADWCVSPNWFCMVMGCTGACTGGTGCGTTTCCCCGAACTG 5 gly C (SEQ ID NO: 9) GTTCTGCATGGTTATG(SEQ ID NO: 158) Myostatin-TN8- IIKFCPWWALFCWDFCACAAATTCTGCCCGTGGTGGGCTCT 5 gly C con10 (SEQ ID NO: 10)GTTCTGCTGGGACTTC (SEQ ID NO: 159) Myostatin-TN8-1 KDLCKMWHWMCKPPAAAGACCTGTGCAAAATGTGGCACTG 5 gly C (SEQ. ID NO: 11) GATGTGCAAACCGCCG(SEQ ID NO: 160) Myostatin-TN8-2 IDKCAIWGWMCPPLATCGACAAATGCGCTATCTGGGGTTG 5 gly C (SEQ ID NO: 12) GATGTGCCCGCCGCTG(SEQ ID NO: 161) Myostatin-TN8-3 WYPCGEFGMWCLNVTGGTACCCGTGCGGTGAATTCGGTAT 5 gly C (SEQ ID NO: 13) GTGGTGCCTGAACGTT(SEQ ID NO: 162) Myostatin-TN8-4 WFTCLWNCDNE TGGTTCACCTGCCTGTGGAACTGCGA5 gly C (SEQ ID NO: 14) CAACGAA (SEQ ID NO: 163) Myostatin-TN8-5HTPCPWFAPLCVEW CACACCCCGTGCCCGTGGTTCGCTCC 5 gly C (SEQ ID NO: 15)GCTGTGCGTTGAATGG (SEQ ID NO: 164) Myostatin-TN8-6 KEWCWRWKWMCKPEAAAGAATGGTGCTGGCGTTGGAAATG 5 gly C (SEQ ID NO: 16) GATGTGCAAACCGGAA(SEQ ID NO: 165) Myostatin-TN8-7 FETCPSWAYFCLDITTCGAAACCTGCCCGTCCTGGGCTTA 5 gly C (SEQ ID NO: 17) CTTCTGCCTGGACATC(SEQ ID NO: 166) Myostatin-TN8-7 FETCPSWAYFCLDITTCGAAACCTGCCCGTCCTGGGCTTA 5 gly N (SEQ ID NO: 17) CTTCTGCCTGGACATC(SEQ ID NO: 167) Myostatin-TN8-8 AYKCEANDWGCWWLGCTTACAAATGCGAAGCTAACGACTG 5 gly C (SEQ ID NO: 18) GGGTTGCTGGTGGCTG(SEQ ID NO: 168) Myostatin-TN8-9 NSWCEDQWHRCWWLAACTCCTGGTGCGAAGACCAGTGGCA 5 gly C (SEQ ID NO: 19) CCGTTGCTGGTGGCTG(SEQ ID NO: 169) Myostatin-TN8-10 WSACYAGIIFWCYDLTGGTCCGCTTGCTACGCTGGTCACTTC 5 gly C (SEQ ID NO: 20) TGGTGCTACGACCTG(SEQ ID NO: 170) Myostatin-TN8-11 ANWCVSPNWFCMVMGCTAACTGGTGCGTTTCCCCGAACTG 5 gly C (SEQ ID NO: 21) GTTCTGCATGGTTATG(SEQ ID NO: 171) Myostatin-TN8-12 WTECYQQEFWCWNLTGGACCGAATGCTACCAGCAGGAATT 5 gly C (SEQ ID NO: 22) CTGGTGCTGGAACCTG(SEQ ID NO: 172) Myostatin-TN8-13 ENTCERWKWMCPPKGAAAACACCTGCGAACGTTGGAAATG 5 gly C (SEQ ID NO: 23) GATGTGCCCGCCGAAA(SEQ ID NO: 173) Myostatin-TN8-14 WLPCHQEGFWCMNFTGGCTGCCGTGCCACCAGGAAGGTTT 5 gly C (SEQ ID NO: 24) CTGGTGCATGAACTTC(SEQ ID NO: 174) Myostatin-TN8-15 STMCSQWIIWMCNPFTCCACCATGTGCTCCCAGTGGCACTG 5 gly C (SEQ ID NO: 25) GATGTGCAACCCGTTC(SEQ ID NO: 175) Myostatin-TN8-16 IFGCHWWDVDCYQFATCTTCGGTTGCCACTGGTGGGACGT 5 gly C (SEQ ID NO: 26) TGACTGCTACCAGTTC(SEQ ID NO: 176) Myostatin-TN8-17 IYGCKWWDIQCYDIATCTACGGTTGCAAATGGTGGGACAT 5 gly C (SEQ ID NO: 27) CCAGTGCTACGACATC(SEQ ID NO: 177) Myostatin-TN8-18 PDWCIDPDWWCKFWCCGGACTGGTGCATCGATCCGGACTG 5 gly C (SEQ ID NO: 28) GTGGTGCAAATTCTGG(SEQ ID NO: 178) Myostatin-TN8-19 QGHCTRWPWMCPPYCAGGGTCACTGCACCCGTTGGCCGTG 5 gly C (SEQ ID NO: 29) GATGTGCCCGCCGTAC(SEQ ID NO: 179) Myostatin-TN8-20 WQECYREGFWCLQTTGGCAGGAATGCTACCGTGAAGGTTT 5 gly C (SEQ ID NO: 30) CTGGTGCCTGCAGACC(SEQ ID NO: 180) Myostatin-TN8-21 WFDCYGPGFKCWSPTGGTTCGACTGCTACGGTCCGGGTTTC 5 gly C (SEQ ID NO: 31) AAATGCTGGTCCCCG(SEQ ID NO: 181) Myostatin-TN8-22 GVRCPKGHLWCLYPGGTGTTCGTTGCCCGAAAGGTCACCT 5 gly C (SEQ ID NO: 32) GTGGTGCCTGTACCCG(SEQ ID NO: 182) Myostatin-TN8-23 HWACGYWPWSCKWVCACTGGGCTTGCGGTTACTGGCCGTG 5 gly C (SEQ ID NO: 33) GTCCTGCAAATGGGTT(SEQ ID NO: 183) Myostatin-TN8-24 GPACHSPWWWCVFGGGTCCGGCTTGCCACTCCCCGTGGTG 5 gly C (SEQ ID NO: 34) GTGGTGCGTTTTCGGT(SEQ ID NO: 184) Myostatin-TN8-25 TTWCISPMWFCSQQACCACCTGGTGCATCTCCCCGATGTG 5 gly C (SEQ ID NO: 35) GTTCTGCTCCCAGCAG(SEQ ID NO: 185) Myostatin-TN8-26 HKFCPPWAIFCWDFCACAAATTCTGCCCGCCGTCGGCTAT 5 gly N (SEQ ID NO: 36) CTTCTGCTGGGACTTC(SEQ ID NO: 186) Myostatin-TN8-27 PDWCVSPRWYCNMWCCGGACTGGTGCGTTTCCCCGCGTTG 5 gly N (SEQ ID NO: 37) GTACTGCAACATGTGG(SEQ ID NO: 187) Myostatin-TN8-28 VWKCHWFGMDCEPTGTTTGGAAATGCCACTGGTTCGGTAT 5 gly N (SEQ ID NO: 38) GGACTGCGAACCGACC(SEQ ID NO: 188) Myostatin-TN8-29 KKHCQIWTWMCAPKAAAAAACACTGCCAGATCTGGACCTG 5 gly N (SEQ ID NO: 39) GATGTGCGCTCCGAAA(SEQ ID NO: 189) Myostatin-TN8-30 WFQCGSTLFWCYNLTGGTTCCAGTGCGGTTCCACCCTGTTC 5 gly N (SEQ ID NO: 40) TGGTGCTACAACCTG(SEQ ID NO: 190) Myostatin-TN8-31 WSPCYDHYFYCYTITGGTCCCCGTGCTACGACCACTACTTC 5 gly N (SEQ ID NO: 41) TACTGCTACACCATC(SEQ ID NO: 191) Myostatin-TN8-32 SWMCGFFKEVCMWVTCCTGGATGTGCGGTTTCTTCAAAGA 5 gly N (SEQ ID NO: 42) AGTTTGCATGTGGGTT(SEQ ID NO: 192) Myostatin-TN8-33 EMLCMIHPVFCNPHGAAATGCTGTGCATGATCCACCCGGT 5 gly N (SEQ ID NO: 43) TTTCTGCAACCCGCAC(SEQ ID NO: 193) Myostatin-TN8-34 LKTCNLWPWMCPPLCTGAAAACCTGCAACCTGTGGCCGTG 5 gly N (SEQ ID NO: 44) GATGTGCCCGCCGCTG(SEQ ID NO: 194) Myostatin-TN8-35 VVGCKWYEAWCYNKGTTGTTGGTTGCAAATGGTACGAAGC 5 gly N (SEQ ID NO: 45) TTGGTGCTACAACAAA(SEQ ID NO: 195) Myostatin-TN8-36 PIHCTQWAWMCPPTCCGATCCACTGCACCCAGTGGGCTTG 5 gly N (SEQ ID NO: 46) GATGTGCCCGCCGACC(SEQ ID NO: 196) Myostatin-TN8-37 DSNCPWYFLSCVIFGACTCCAACTGCCCGTGGTACTTCCT 5 gly N (SEQ ID NO: 47) GTCCTGCGTTATCTTC(SEQ ID NO: 197) Myostatin-TN8-38 HIWCNLAMMKCVEMCACATCTGGTGCAACCTGGCTATGAT 5 gly N (SEQ ID NO: 48) GAAATGCGTTGAAATG(SEQ ID NO: 198) Myostatin-TN8-39 NLQCIYFLGKCIYFAACCTGCAGTGCATCTACTTCCTGGG 5 gly N (SEQ ID NO: 49) TAAATGCATCTACTTC(SEQ ID NO: 199) Myostatin-TN8-40 AWRCMWFSDVCTPGGCTTGGCGTTGCATGTGGTTCTCCGAC 5 gly N (SEQ ID NO: 50) GTTTGCACCCCGGGT(SEQ ID NO: 200) Myostatin-TN8-41 WFRCFLDADWCTSVTGGTTTCGTTGTTTTCTTGATGCTGAT 5 gly N (SEQ ID NO: 51) TGGTGTACTTCTGTT(SEQ ID NO: 201) Myostatin-TN8-42 EKICQMWSWMCAPPGAAAAAATTTGTCAAATGTGGTCTTG 5 gly N (SEQ ID NO: 52) GATGTGTGCTCCACCA(SEQ ID NO: 202) Myostatin-TN8-43 WFYCHLNKSECTEPTGGTTTTATTGTCATCTTAATAAATCT 5 gly N (SEQ ID NO: 53) GAATGTACTGAACCA(SEQ ID NO: 203) Myostatin-TN8-44 FWRCAIGIDKCKRVTTTTGGCGTTGTGCTATTGGTATTGAT 5 gly N (SEQ ID NO: 54) AAATGTAAACGTGTT(SEQ ID NO: 204) Myostatin-TN8-45 NLGCKWYEVWCFTYAATCTTGGTTGTAAATGGTATGAAGT 5 gly N (SEQ ID NO: 55) TTGGTGTTTTACTTAT(SEQ ID NO: 205) Myostatin-TN8-46 IDLCNMWDGMCYPPATTGATCTTTGTAATATGTGGGATGGT 5 gly N (SEQ ID NO: 56) ATGTGTTATCCACCA(SEQ ID NO: 206) Myostatin-TN8-47 EMPCNIWGWMCPPVGAAATGCCATGTAATATTTGGGGTTG 5 gly N (SEQ ID NO: 57) GATGTGTCCACCAGTT(SEQ ID NO: 207) Myostatin-TN12-1 WFRCVLTGIVDWSECFTGGTTCCGTTGCGTTCTGACCGGTATC 5 gly N GL GTTGACTGGTCCGAATGCTTCGGTCT(SEQ ID NO: 58) G (SEQ ID NO: 208) Myostatin-TN12-2 GFSCTFGLDEFYVDCSPGGTTTCTCCTGCACCTTCGGTCTGGAC 5 gly N F GAATTCTACGTTGACTGCTCCCCGTTC(SEQ ID NO: 59) (SEQ ID NO: 209) Myostatin-TN12-3 LPWCHDQVNADWGFCCTGCCGTGGTGCCACGACCAGGTTAA 5 gly N MLW CGCTGACTGGGGTTTCTGCATGCTGT(SEQ ID NO: 60) GG (SEQ ID NO: 210) Myostatin-TN12-4 YPTCSEKFWIYGQTCVTACCCGACCTGCTCCGAAAAATTCTG 5 gly N LW GATCTACGGTCAGACCTGCGTTCTGT(SEQ ID NO: 61) GG (SEQ ID NO: 211) Myostatin-TN12-5 LGPCPIHHGPWPQYCVCTGGGTCCGTGCCCGATCCACCACGG 5 gly N YW TCCGTGGCCGCAGTACTGCGTTTACT(SEQ ID NO: 62) GG (SEQ ID NO: 212) Myostatin-TN12-6 PFPCETIIQISWLGIICLSCCGTTCCCGTGCGAAACCCACCAGAT 5 gly N F CTCCTGGCTGGGTCACTGCCTGTCCTT(SEQ ID NO: 63) C (SEQ ID NO: 213) Myostatin-TN12-7 HWGCEDLMWSWHPLCCACTGGGGTTGCGAAGACCTGATGTG 5 gly N RRP GTCCTGGCACCCGCTGTGCCGTCGTC(SEQ ID NO: 64) CG (SEQ ID NO: 214) Myostatin-TN12-8 LPLCDADMMPTTGFCVCTGCCGCTGTGCGACGCTGACATGAT 5 gly N AY GCCGACCATCGGTTTCTGCGTTGCTTA(SEQ ID NO: 65) C (SEQ ID NO: 215) Myostatin-TN12-9 SHWCETTFWMNYAKCTCCCACTGGTGCGAAACCACCTTCTG 5 gly N VHA GATGAACTACGCTAAATGCGTTCACG(SEQ ID NO: 66) CT (SEQ ID NO: 216) Myostatin-TN12-10 LPKCTHVPFDQGGFCLCTGCCGAAATGCACCCACGTTCCGTT 5 gly N WY CGACCAGGGTGGTTTCTGCCTGTGGT(SEQ ID NO: 67) AC (SEQ ID NO: 217) Myostatin-TN12-11 FSSCWSPVSRQDMFCVTTCTCCTCCTGCTGGTCCCCGGTTTCC 5 gly N FY CGTCAGGACATGTTCTGCGTTTTCTAC(SEQ ID NO: 68) (SEQ ID NO: 218) Myostatin-TN12-13 SHKCEYSGWLQPLCYRTCCCACAAATGCGAATACTCCGGTTG 5 gly N P GCTGCAGCCGCTGTGCTACCGTCCG(SEQ ID NO: 69) (SEQ ID NO: 219) Myostatin-TN12-14 PWWCQDNYVQHMLHCCGTGGTGGTGCCAGGACAACTACGT 5 gly N CDSP TCAGCACATGCTGCACTGCGACTCCC(SEQ ID NO: 70) CG (SEQ ID NO: 220) Myostatin-TN12-15 WFRCMLMNSFDAFQCTGGTTCCGTTGCATGCTGATGAACTCC 5 gly N VSY TTCGACGCTTTCCAGTGCGTTTCCTAC(SEQ ID NO: 71) (SEQ ID NO: 221) Myostatin-TN12-16 PDACRDQPWYMFMGCCCGGACGCTTGCCGTGACCAGCCGTG 5 gly N MLG GTACATGTTCATGGGTTGCATGCTGG(SEQ ID NO: 72) GT (SEQ ID NO: 222) Myostatin-TN12-17 FLACFVEFELCFDSTTCCTGGCTTGCTTCGTTGAATTCGAA 5 gly N (SEQ ID NO: 73) CTGTGCTTCGACTCC(SEQ ID NO: 223) Myostatin-TN12-18 SAYCIITESDPYVLCVPTCCGCTTACTGCATCATCACCGAATCC 5 gly N L GACCCGTACGTTCTGTGCGTTCCGCTG(SEQ ID NO: 74) (SEQ ID NO: 224) Myostatin-TN12-19 PSICESYSTMWLPMCQCCGTCCATCTGCGAATCCTACTCCACC 5 gly N HN ATGTGGCTGCCGATGTGCCAGCACAA(SEQ ID NO: 75) C (SEQ ID NO: 225) Myostatin-TN12-20 WLDCHDDSWAWTKMTGGCTGGACTGCCACGACGACTCCTG 5 gly N CRSH GGCTTGGACCAAAATGTGCCGTTCCC(SEQ ID NO: 76) AC (SEQ ID NO: 226) Myostatin-TN12-21 YLNCVMMNTSPFVECTACCTGAACTGCGTTATGATGAACAC 5 gly N VFN CTCCCCGTTCGTTGAATGCGTTTTCAA(SEQ ID NO: 77) C (SEQ ID NO: 227) Myostatin-TN12-22 YPWCDGFMIQQGITCMTACCCGTGGTGCGACGGTTTCATGAT 5 gly N FY CCAGCAGGGTATCACCTGCATGTTCT(SEQ ID NO: 78) AC (SEQ ID NO: 228) Myostatin-TN12-23 FDYCTWLNGFKDWKCTTCGACTACTGCACCTGGCTGAACGG 5 gly N WSR TTTCAAAGACTGGAAATGCTGGTCCC(SEQ ID NO: 79) GT (SEQ ID NO: 229) Myostatin-TN12-24 LPLCNLKEISHVQACVLCTGCCGCTGTGCAACCTGAAAGAAAT 5 gly N F CTCCCACGTTCAGGCTTGCGTTCTGTT(SEQ ID NO: 80) C (SEQ ID NO: 230) Myostatin-TN12-25 SPECAFARWLGIEQCQTCCCCGGAATGCGCTTTCGCTCGTTGG 5 gly N RD CTGGGTATCGAACAGTGCCAGCGTGA(SEQ ID NO: 81) C (SEQ ID NO: 231) Myostatin-TN12-26 YPQCFNLIILLEWTECDTACCCGCAGTGCTTCAACCTGCACCT 5 gly N WF GCTGGAATGGACCGAATGCGACTGGT(SEQ ID NO: 82) TC (SEQ ID NO: 232) Myostatin-TN12-27 RWRCEIYDSEFLPKCWCGTTGGCGTTGCGAAATCTACGACTC 5 gly N FF CGAATTCCTGCCGAAATGCTGGTTCTT(SEQ ID NO: 83) C (SEQ ID NO: 233) Myostatin-TN12-28 LVGCDNVWHRCKLFCTGGTTGGTTGCGACAACGTTTGGCA 5 gly N (SEQ ID NO: 84) CCGTTGCAAACTGTTC(SEQ ID NO: 234) Myostatin-TN12-29 AGWCHVWGEMFGMGGCTGGTTGGTGCCACGTTTGGGGTGA 5 gly N CSAL AATGTTCGGTATGGGTTGCTCCGCTCT(SEQ ID NO: 85) G (SEQ ID NO: 235) Myostatin-TN12-30 HHECEWMARWMSLDCACCACGAATGCGAATGGATGGCTCG 5 gly N CVGL TTGGATGTCCCTGGACTGCGTTGGTCT(SEQ ID NO: 86) G (SEQ ID NO: 236) Myostatin-TN12-31 FPMCGIAGMKDFDFCVTTCCCGATGTGCGGTATCGCTGGTAT 5 gly N WY GAAAGACTTCGACTTCTGCGTTTGGT(SEQ ID NO: 87) AC (SEQ ID NO: 237) Myostatin-TN12-32 RDDCTFWPEWLWKLCCGTGATGATTGTACTTTTTGGCCAGAA 5 gly N ERP TGGCTTTGGAAACTTTGTGAACGTCC(SEQ ID NO: 88) A (SEQ ID NO: 238) Myostatin-TN12-33 YNFCSYLFGVSKEACQTATAATTTTTGTTCTTATCTTTTTGGTG 5 gly N LP TTTCTAAAGAAGCTTGTCAACTTCCA(SEQ ID NO: 89) (SEQ ID NO: 239) Myostatin-TN12-34 AHWCEQGPWRYGNICGCTCATTGGTGTGAACAAGGTCCATG 5 gly N MAY GCGTTATGGTAATATTTGTATGGCTTA C(SEQ ID NO: 90) T (SEQ ID NO: 240) Myostatin-TN12-35 NLVCGKISAWGDEACAAATCTTGTTTGTGGTAAAATTTCTGCT 5 gly N RA TGGGGTGATGAAGCTTGTGCTCGTGC(SEQ ID NO: 91) T (SEQ ID NO: 241) Myostatin-TN12-36 HNVCTIMGPSMKWFCCATAATGTTTGTACTATTATGGGTCCA 5 gly N WND TCTATGAAATGGTTTTGTTGGAATGAT C(SEQ ID NO: 92) (SEQ ID NO: 242) Myostatin-TN12-37 NDLCAMWGWRNTIWCAATGATCTTTGTGCTATGTGGGGTTGG 5 gly N QNS CGTAATACTATTTGGTGTCAAAATTCT C(SEQ ID NO: 93) (SEQ ID NO: 243) Myostatin-TN12-38 PPFCQNDNDMLQSLCKCCACCATTTTGTCAAAATGATAATGA 5 gly N LL TATGCTTCAATCTCTTTGTAAACTTCT(SEQ ID NO: 94) T (SEQ ID NO: 244) Myostatin-TN12-39 WYDCNVPNELLSGLCRTGGTATGATTGTAATGTTCCAAATGA 5 gly N LF ACTTCTTTCTGGTCTTTGTCGTCTTTTT(SEQ ID NO: 95) (SEQ ID NO: 245) Myostatin-TN12-40 YGDCDQNHWMWPFTCTATGGTGATTGTGATCAAAATCATTG 5 gly N LSL GATGTGGCCATTTACTTGTCTTTCTCT C(SEQ ID NO: 96) T (SEQ ID NO: 246) Myostatin-TN12-41 GWMCHFDLHDWGATGGTTGGATGTGTCATTTTGATCTTCAT 5 gly N CQPD GATTGGGGTGCTACTTGTCAACCAGA(SEQ ID NO: 97) T (SEQ ID NO: 247) Myostatin-TN12-42 YFHCMFGGHEFEVHCETATTTTCATTGTATGTTTGGTGGTCAT 5 gly N SF GAATTTGAAGTTCATTGTGAATCTTTT C(SEQ ID NO: 98) (SEQ ID NO: 248) Myostatin-TN12-43 AYWCWHGQCVRFGCTTATTGGTGTTGGCATGGTCAATGT 5 gly N (SEQ ID NO: 99) CTTCGTTTT(SEQ ID NO: 249) Myostatin-Linear-1 SEHWTFTDWDGNEWTCCGAACACTGGACCTTCACCGACTG 5 gly N WVRPF GGACGGTAACGAATGGTGGGTTCGTC(SEQ ID NO: 100) CGTTC (SEQ ID NO: 250) Myostatin-Linear-2MEMLDSLFELLKDMVP ATGGAAATGCTGGACTCCCTGTTCGA 5 gly N ISKAACTGCTGAAAGACATGGTTCCGATCT (SEQ ID NO: 101) CCAAAGCT (SEQ ID NO: 251)Myostatin-Linear-3 SPPEEALMEWLGWQY TCCCCGCCGGAAGAAGCTCTGATGGA 5 gly NGKFT ATGGCTGGGTTGGCAGTACGGTAAAT (SEQ ID NO: 102) TCACC (SEQ ID NO: 252)Myostatin-Linear-4 SPENLLNDLYILMTKQ TCCCCGGAAAACCTGCTGAACGACCT 5 gly NEWYG GTACATCCTGATGACCAAACAGGAAT (SEQ ID NO: 103) GGTACGGT(SEQ ID NO: 253) Myostatin-Linear-5 FHWEEGIPFHVVTPYSTTCCACTGGGAAGAAGGTATCCCGTT 5 gly N YDRM CCACGTTGTTACCCCGTACTCCTACGA(SEQ ID NO:104) CCGTATG (SEQ ID NO: 254) Myostatin-Linear-6KRLLEQFMNDLAELVS AAACGTCTGCTGGAACAGTTCATGAA 5 gly N GIISCGACCTGGCTGAACTGGTTTCCGGTC (SEQ ID NO: 105) ACTCC (SEQ ID NO: 255)Myostatin-Linear-7 DTRDALFQEFYEFVRS GACACCCGTGACGCTCTGTTCCAGGA 5 gly NRLVI ATTCTACGAATTCGTTCGTTCCCGTCT (SEQ ID NO: 106) GGTTATC(SEQ ID NO: 256) Myostatin-Linear-8 RMSAAPRPLTYRDIMDCGTATGTCCGCTGCTCCGCGTCCGCTG 5 gly N QYWH ACCTACCGTGACATCATGGACCAGTA(SEQ ID NO: 107) CTGGCAC (SEQ ID NO: 257) Myostatin-Linear-9NDKAIIFFEMFMFDVII AACGACAAAGCTCACTTCTTCGAAAT 5 gly N NFVESGTTCATGTTCGACGTTCACAACTTCGT (SEQ ID NO: 108) TGAATCC (SEQ ID NO: 258)Myostatin-Linear-10 QTQAQKIDGLWELLQS CAGACCCAGGCTCAGAAAATCGACGG 5 gly NIRNQ TCTGTGGGAACTGCTGCAGTCCATCC (SEQ ID NO: 109) GTAACCAG(SEQ ID NO: 259) Myostatin-Linear-11 MLSEFEEFLGNLVHRQATGCTGTCCGAATTCGAAGAATTCCT 5 gly N EA GGGTAACCTGGTTCACCGTCAGGAAG(SEQ ID NO: 110) CT (SEQ ID NO: 260) Myostatin-Linear-12 YTPKMGSEWTSFWHNTACACCCCGAAAATGGGTTCCGAATG 5 gly N RIHYL GACCTCCTTCTGGCACAACCGTATCC(SEQ ID NO: 111) ACTACCTG (SEQ ID NO: 261) Myostatin-Linear-13LNDTLLRELKMVLNSL CTGAACGACACCCTGCTGCGTGAACT 5 gly N SDMKGAAAATGGTTCTGAACTCCCTGTCCG (SEQ ID NO: 112) ACATGAAA (SEQ ID NO: 262)Myostatin-Linear-14 FDVERDLMRWLEGFM TTCGACGTTGAACGTGACCTGATGCG 5 gly NQSAAT TTGGCTGGAAGGTTTCATGCAGTCCG (SEQ ID NO: 113) CTGCTACC(SEQ ID NO: 263) Myostatin-Linear-15 HHGWNYLRKGSAPQWCACCACGGTTGGAACTACCTGCGTAA 5 gly N FEAWV AGGTTCCGCTCCGCAGTGGTTCGAAG(SEQ ID NO: 114) CTTGGGTT (SEQ ID NO: 264) Myostatin-Linear-16VESLHQLQMWLDQKL GTTGAATCCCTGCACCAGCTGCAGAT 5 gly N ASGPHGTGGCTGGACCAGAAACTGGCTTCCG (SEQ ID NO: 115) GTCCGCAC (SEQ ID NO: 265)Myostatin-Linear-17 RATLLKDFWQLVEGY CGTGCTACCCTGCTGAAAGACTTCTG 5 gly NGDN GCAGCTGGTTGAAGGTTACGGTGACA (SEQ ID NO: 116) AC (SEQ ID NO: 266)Myostatin-Linear-18 EELLREFYRFVSAFDY GAAGAACTGCTGCGTGAATTCTACCG 5 gly N(SEQ ID NO: 117) TTTCGTTTCCGCTTTCGACTAC (SEQ ID NO: 267)Myostatin-Linear-19 GLLDEFSHFIAEQFYQ GGTCTGCTGGACGAATTCTCCCACTTC 5 gly NMPGG ATCGCTGAACAGTTCTACCAGATGCC (SEQ ID NO: 118) GGGTGGT(SEQ ID NO: 268) Myostatin-Linear-20 YREMSMLEGLLDVLERTACCGTGAAATGTCCATGCTGGAAGG 5 gly N LQHY TCTGCTGGACGTTCTGGAACGTCTGC(SEQ ID NO: 119) AGCACTAC (SEQ ID NO: 269) Myostatin-Linear-21IINSSQMLLSELIMLVG CACAACTCCTCCCAGATGCTGCTGTC 5 gly N SMMQCGAACTGATCATGCTGGTTGGTTCCA (SEQ ID NO: 120) TGATGCAG (SEQ ID NO: 270)Myostatin-Linear-22 WREHFLNSDYIRDKLI TGGCGTGAACACTTCCTGAACTCCGA 5 gly NAIDG CTACATCCGTGACAAACTGATCGCTA (SEQ ID NO: 121) TCGACGGT(SEQ ID NO: 271) Myostatin-Linear-23 QFPFYVFDDLPAQLEYCAGTTCCCGTTCTACGTTTTCGACGAC 5 gly N WIA CTGCCGGCTCAGCTGGAATACTGGAT(SEQ ID NO: 122) CGCT (SEQ ID NO: 272) Myostatin-Linear-24EFFHWLHNHRSEVNH GAATTCTTCCACTGGCTGCACAACCA 5 gly N WLDMNCCGTTCCGAAGTTAACCACTGGCTGG (SEQ ID NO: 123) ACATGAAC (SEQ ID NO: 273)Myostatin-Linear-25 EALFQNFFRDVLTLSER GAAGCTCTTTTTCAAAATTTTTTTCGT 5 glyN EY GATGTTCTTACTCTTTCTGAACGTGAA C (SEQ ID NO: 124) TAT (SEQ ID NO: 274)Myostatin-Linear- QYWEQQWMTYFRENG CAATATTGGGAACAACAATGGATGAC 5 gly N 26LHVQY TTATTTTCGTGAAAATGGTCTTCATGT (SEQ ID NO: 125) TCAATAT(SEQ ID NO: 275) Myostatin-Linear-27 NQRMMLEDLWRIMTPAATCAACGTATGATGCTTGAAGATCT 5 gly N MFGRS TTGGCGTATTATGACTCCAATGTTTGG C(SEQ ID NO: 126) TCGTTCT (SEQ ID NO: 276) Myostatin-Linear-29FLDELKAELSRHYALD TTTCTTGATGAACTTAAAGCTGAACTT 5 gly N DLDETCTCGTCATTATGCTCTTGATGATCTT (SEQ ID NO: 127) GATGAA (SEQ ID NO: 277)Myostatin-Linear-30 GKLIEGLLNELMQLETF GGTAAACTTATTGAAGGTCTTCTTAAT 5 glyN MPD GAACTTATGCAACTTGAAACTTTTATG C (SEQ ID NO: 128) CCAGAT(SEQ ID NO: 278) Myostatin-Linear-31 ILLLDEYKKDWKSWFATTCTTCTTCTTGATGAATATAAAAAA 5 gly N (SEQ ID NO: 129) GATTGGAAATCTTGGTTT(SEQ ID NO: 279) Myostatin-2XTN8- QGHCTRWPWMCPPYGCAGGGCCACTGTACTCGCTGGCCGTG 1k N 19 kc SGSATGGSGSTASSGSGGATGTGCCCGCCGTACGGTTCTGGTT SATGQGHCTRWPWMC CCGCTACCGGTGGTTCTGGTTCCACTGPPY CTTCTTCTGGTTCCGGTTCTGCTACTG (SEQ ID NO: 130)GTCAGGGTCACTGCACTCGTTGGCCA TGGATGTGTCCACCGTAT (SEQ ID NO: 280)Myostatin-2XTN8- WYPCYEGHFWCYDLG TGGTATCCGTGTTATGAGGGTCACTTC 5 gly CCON6 SGSTASSGSGSATGWY TGGTGCTACGATCTGGGTTCTGGTTCC PCYEGHFWCYDLACTGCTTCTTCTGGTTCCGGTTCCGCT (SEQ ID NO: 131) ACTGGTTGGTACCCGTGCTACGAAGGTCACTTTTGGTGTTATGATCTG (SEQ ID NO: 281) Myostatin-2XTN8-5HTPCPWFAPLCVEWGS CACACTCCGTGTCCGTGGTTTGCTCCG 1k C kc GSATGGSGSTASSGSGSCTGTGCGTTGAATGGGGTTCTGGTTCC ATGHTPCPWFAPLCVE GCTACTGGTGGTTCCGGTTCCACTGCTW TCTTCTGGTTCCGGTTCTGCAACTGGT (SEQ ID NO: 132)CACACCCCGTGCCCGTGGTTTGCACC GCTGTGTGTAGAGTGG (SEQ ID NO: 282)Myostatin-2XTN8- PDWCIDPDWWCKFWG CCGGATTGGTGTATCGACCCGGACTG 1k C 18 kcSGSATGGSGSTASSGSG GTGGTGCAAATTCTGGGGTTCTGGTTC SATGPDWCIDPDWWCCGCTACCGGTGGTTCCGGTTCCACTG KFW CTTCTTCTGGTTCCGGTTCTGCAACTG(SEQ ID NO: 133) GTCCGGACTGGTGCATCGACCCGGAT TGGTGGTGTAAATTTTGG(SEQ ID NO: 283) Myostatin-2XTN8- ANWCVSPNWFCMVMCCGGATTGGTGTATCGACCCGGACTG 1k C 11 kc GSGSATGGSGSTASSGSGTGGTGCAAATTCTGGGGTTCTGGTTC GSATGANWCVSPNWF CGCTACCGGTGGTTCCGGTTCCACTGCMVM CTTCTTCTGGTTCCGGTTCTGCAACTG (SEQ ID NO: 134)GTCCGGACTGGTGCATCGACCCGGAT TGGTGGTGTAAATTTTGG (SEQ ID NO: 284)Myostatin-2XTN8- PDWCIDPDWWCKFWG ACCACTTGGTGCATCTCTCCGATGTG 1k C 25 kcSGSATGGSGSTASSGSG GTTCTGCTCTCAGCAGGGTTCTGGTTC SATGPDWCIDPDWWCCACTGCTTCTTCTGGTTCCGGTTCTGC KFW AACTGGTACTACTTGGTGTATCTCTCC(SEQ ID NO: 135) AATGTGGTTTTGTTCTCAGCAA (SEQ ID NO: 285)Myostatin-2XTN8- HWACGYWPWSCKWV CACTGGGCATGTGGCTATTGGCCGTG 1k C 23 kcGSGSATGGSGSTASSGS GTCCTGCAAATGGGTTGGTTCTGGTTC GSATGHWACGYWPWSCGCTACCGGTGGTTCCGGTTCCACTG CKWV CTTCTTCTGGTTCCGGTTCTGCAACTG(SEQ ID NO: 136) GTCACTGGGCTTGCGGTTACTGGCCG TGGTCTTGTAAATGGGTT(SEQ ID NO: 286) Myostatin-TN8-29- KKHCQIWTWMCAPKGAAAAAACACTGTCAGATCTGGACTTG 1k C 19 kc SGSATGGSGSTASSGSGGATGTGCGCTCCGAAAGGTTCTGGTT SATGQGHCTRWPWMC CCGCTACCGGTGGTTCTGGTTCCACTGPPY CTTCTTCTGGTTCCGGTTCCGCTACTG (SEQ ID NO: 137)GTCAGGGTCACTGCACTCGTTGGCCA TGGATGTGTCCGCCGTAT (SEQ ID NO: 287)Myostatin-TN8-19- QGHCTRWPWMCPPYG CAGGGTCACTGCACCCGTTGGCCGTG 1k C 29 kcSGSATGGSGSTASSGSG GATGTGCCCGCCGTACGGTTCTGGTT SATGKKHCQIWTWMCCCGCTACCGGTGGTTCTGGTTCCACTG APK CTTCTTCTGGTTCCGGTTCTGCTACTG(SEQ ID NO: 138) GTAAAAAACACTGCCAGATCTGGACT TGGATGTGCGCTCCGAAA(SEQ ID NO: 288) Myostatin-TN8-29- KKHCQIWTWMCAPKGAAAAAACACTGTCAGATCTGGACTTG 1k N 19 kn SGSATGGSGSTASSGSGGATGTGCGCTCCGAAAGGTTCTGGTT SATGQGHCTRWPWMC CCGCTACCGGTGGTTCTGGTTCCACTGPPY CTTCTTCTGGTTCCGGTTCCGCTACTG (SEQ ID NO: 139)GTCAGGGTCACTGCACTCGTTGGCCA TGGATGTGTCCGCCGTAT (SEQ ID NO: 289)Myostatin-TN8-29- KKHCQIWTWMCAPKG AAAAAACACTGCCAGATCTGGACTTG 8 gly C19-8g GGGGGGGQGHCTRWP GATGTGCGCTCCGAAAGGTGGTGGTG WMCPPYGTGGTGGCGGTGGCCAGGGTCACTGC (SEQ ID NO: 140) ACCCGTTGGCCGTGGATGTGTCCGCCGTAT (SEQ ID NO: 290) Myostatin-TN8-19- QGHCTRWPWMCPPYGCAGGGTCACTGCACCCGTTGGCCGTG 6 gly C 29-6gc GGGGGKKHCQIWTWMGATGTGCCCGCCGTACGGTGGTGGTG CAPK GTGGTGGCAAAAAACACTGCCAGATC(SEQ ID NO: 141) TGGACTTGGATGTGCGCTCCGAAA (SEQ ID NO: 291)

Example 3 In Vitro Assays

C2C12 Cell Based Myostatin Activity Assay

This assay demonstrates the myostatin neutralizing capability of theinhibitor being tested by measuring the extent that binding of myostatinto its receptor is inhibited.

A myostatin-responsive reporter cell line was generated by transfectionof C2C12 myoblast cells (ATCC No: CRL-1772) with a pMARE-luc construct.The pMARE-luc construct was made by cloning twelve repeats of the CAGAsequence, representing the myostatin/activin response elements (Dennleret al. EMBO 17: 3091-3100 (1998)) into a pLuc-MCS reporter vector(Stratagene cat #219087) upstream of the TATA box. The myoblast C2C12cells naturally express myostatin/activin receptors on its cell surface.When myostatin binds the cell receptors, the Smad pathway is activated,and phosphorylated Smad binds to the response element (Maciasi-Silva etal. Cell 87:1215 (1996)), resulting in the expression of the lucerasegene. Luciferase activity is then measured using a commercial luciferasereporter assay kit (cat #E4550, Promega, Madison, Wis.) according tomanufacturer's protocol. A stable line of C2C12 cells that had beentransfected with pMARE-luc (C2C12/pMARE clone #44) was used to measuremyostatin activity according to the following procedure.

Equal numbers of the reporter cells (C2C12/pMARE clone #44) were platedinto 96 well cultures. A first round screening using two dilutions ofpeptibodies was performed with the myostatin concentration fixed at 4nM. Recombinant mature myostatin was pre-incubated for 2 hours at roomtemperature with peptibodies at 40 nM and 400 nM respectively. Thereporter cell culture was treated with the myostatin with or withoutpeptibodies for six hours. Myostatin activity was measured bydetermining the luciferase activity in the treated cultures. This assaywas used to initially identify peptibody hits that inhibited themyostatin signaling activity in the reporter assay. Subsequently, a ninepoint titration curve was generated with the myostatin concentrationfixed at 4 nM. The myostatin was preincubated with each of the followingnine concentrations of peptibodies: 0.04 mM, 0.4 nM, 4 nM, 20 nM, 40 nM,200 nM, 400 nM, 2 uM and 4 uM for two hours before adding the mixture tothe reporter cell culture. The IC₅₀ values for a number of exemplarypeptibodies are provided in Table III, and for affinity maturedpeptibodies, in Table VIII.

BIAcore® Assay

An affinity analysis of each candidate myostatin peptibody was performedon a BIAcore® 3000 (Biacore, Inc., Piscataway, N.J.), apparatus usingsensor chip CM5, and 0.005 percent P20 surfactant (Biacore, Inc.) asrunning buffer. Recombinant mature myostatin protein was immobilized toa research grade CM5 sensor chip (Biacore, Inc.) via primary aminegroups using the Amine Coupling Kit (Biacore, Inc.) according to themanufacturer's suggested protocol.

Direct binding assays were used to screen and rank the peptibodies inorder of their ability to bind to immobilized myostatin. Binding assayswere carried by injection of two concentrations (40 and 400 nM) of eachcandidate myostatin-binding peptibody to the immobilized myostatinsurface at a flow rate of 50 μl/min for 3 minutes. After a dissociationtime of 3 minutes, the surface was regenerated. Binding curves werecompared qualitatively for binding signal intensity, as well as fordissociation rates. Peptibody binding kinetic parameters including k_(a)(association rate constant), k_(d) (dissociation rate constant) andK_(D) (dissociation equilibrium constant) were determined using the BTAevaluation 3.1 computer program (Biacore, Inc.). The lower thedissociation equilibrium constants (expressed in nM), the greater theaffinity of the peptibody for myostatin. Examples of peptibody K_(D)values are shown in Table III and Table VI for affinity-maturedpeptibodies below.

Blocking Assay on ActRIIB/Fc Surface

Blocking assays were carried out using immobilized ActRIIB/Fc (R&DSystems, Minneapolis, Minn.) and myostatin in the presence and absenceof peptibodies with the BIAcore® assay system. Assays were used toclassify peptibodies as non-neutralizing (those which did not preventmyostatin binding to ActRIIB/Fc) or neutralizing (those that preventedmyostatin binding to ActRIIB/Fc). Baseline myostatin-ActRIIB/Fc bindingwas first determined in the absence of any peptibody.

For early screening studies, peptibodies were diluted to 4 nM, 40 nM,and 400 nM in sample buffer and incubated with 4 nM myostatin (alsodiluted in sample buffer). The peptibody: ligand mixtures were allowedto reach equilibrium at room temperature (at least 5 hours) and thenwere injected over the immobilized ActRIIB/Fc surface for 20 to 30minutes at a flow rate of 10 uL/min. An increased binding response (overcontrol binding with no peptibody) indicated that peptibody binding tomyostatin was non-neutralizing. A decreased binding response (comparedto the control) indicated that peptibody binding to myostatin blockedthe binding of myostatin to ActRIIB/Fc. Selected peptibodies werefurther characterized using the blocking assay of a full concentrationseries in order to derive IC₅₀ values (for neutralizing peptibodies) orEC₅₀ (for non-neutralizing peptibodies). The peptibody samples wereserially diluted from 200 nM to 0.05 mM in sample buffer and incubatedwith 4 mM myostatin at room temperature to reach equilibrium (minimum offive hours) before injected over the immobilized ActRIIB/Fc surface for20 to 30 minutes at a flow rate of 10 uL/min. Following the sampleinjection, bound ligand was allowed to dissociate from the receptor for3 minutes. Plotting the binding signal vrs. peptibody concentration, theIC₅₀ values for each peptibody in the presence of 4 nM myostatin werecalculated. It was found, for example, that the peptibodies TN8-19, L2and L17 inhibit myostatin activity in cell-based assay, but binding ofTN-8-19 does not block myostatin/ActRIIB/Fc interactions, indicatingthat TN-8-19 binds to a different epitope than that observed for theother two peptibodies.

Epitope Binning for Peptibodies

A purified peptibody was immobilized on a BIAcore chip to capturemyostatin before injection of a second peptibody, and the amount ofsecondary peptibody bound to the captured myostatin was determined Onlypeptibodies with distinct epitopes will bind to the captured myostatin,thus enabling the binning of peptibodies with similar or distinctepitope binding properties. For example, it was shown that peptibodiesTN8-19 and L23 bind to different epitopes on myostatin.

Selectivity Assays

These assays were performed using BIAcore® technology, to determine theselectivity of binding of the peptibodies to other TGFβ family members.ActRIIB/Fc, TGFβRII/Fc and BMPR-1A/Fc (all obtained from R & D Systems,Minneapolis, Minn.) were covalently coupled to research grade sensorchips according to manufacturer's suggested protocol. Because BIAcoreassays detects changes in the refractive index, the difference betweenthe response detected with injection over the immobilized receptorsurfaces compared with the response detected with injection over thecontrol surface in the absence of any peptibody represents the actualbinding of Activin A, TGFβ1, TGFβ3, and BMP4 to the receptors,respectively. With pre-incubation of peptibodies and TGFβ molecules, achange (increase or decrease) in binding response indicates peptibodybinding to the TGFβ family of molecules. The peptibodies of the presentinvention all bind to myostatin but not to Activin A, TGFβ1, TGFβ3, orBMP4.

KinEx A™ Equilibrium Assays

Solution-based equilibrium-binding assays using KinExA™ technology(Sapidyne Instruments, Inc.) were used to determine the dissociationequilibrium (K_(D)) of myostatin binding to peptibody molecules. Thissolution-based assay is considered to be more sensitive than the BIAcoreassay in some instances. Reacti-Gel™ 6× was pre-coated with about 50ug/ml myostatin for over-night, and then blocked with BSA. 30 pM and 100pM of peptibody samples were incubated with various concentrations (0.5pM to 5 nM) of myostatin in sample buffer at room temperature for 8hours before being run through the myostatin-coated beads. The amount ofthe bead-bound peptibody was quantified by fluorescent (Cy5) labeledgoat anti-human-Fc antibody at 1 mg/ml in superblock. The binding signalis proportional to the concentration of free peptibody at equilibriumwith a given myostatin concentration. K_(D) was obtained from thenonlinear regression of the competition curves using a dual-curveone-site homogeneous binding model provided in the KinEx A™ software(Sapidyne Instruments, Inc.).

Example 4 Comparison of Myostatin Inhibitors

The ability of three exemplary first-round peptibodies to bind to(K_(D)) and inhibit (IC₅₀) were compared with the K_(D) and IC₅₀ valuesobtained for the soluble receptor fusion protein actRIIB/Fc (R &DSystems, Inc., Minneapolis, Minn.). The IC₅₀ values were determinedusing the pMARE luc cell-based assay described in Example 3 and theK_(D) values were determined using the Biacore® assay described inExample 3.

TABLE III Inhibitor IC₅₀ (nM) K_(D) (nM) ActRIIB/Fc ~83 ~7 2xTN8-19-kc~9 ~2 TN8-19 ~23 ~2 TN8-29 ~26 ~60 TN12-34 ~30 — Linear-20 ~11 —

The peptibodies have an IC₅₀ that is improved over the receptor/Fcinhibitor and binding affinities which are comparable in two peptibodieswith the receptor/Fc.

Example 5 Comparison of Ability of Peptide and Peptibody to InhibitMyostatin

The following peptide sequence: QGHCTRWPWMCPPY (TN8-19) (SEQ ID NO: 33)was used to construct the corresponding peptibody TN8-19(pb) accordingto the procedure described in Example 2 above. Both the peptide aloneand the peptibody were screened for myostatin inhibiting activity usingthe C2C12 based assay described in Example 3 above. It can be seen fromFIG. 1 the IC₅₀ (effective concentration for fifty percent inhibition ofmyostatin) for the peptibody is significantly less than that of thepeptide, and thus the ability of the peptide to inhibit myostatinactivity has been substantially improved by placing it in the peptibodyconfiguration.

Example 6 Generation of Affinity-Matured Peptides and Peptibodies

Several of the first round peptides used for peptibody generation werechosen for affinity maturation. The selected peptides included thefollowing: the cysteine constrained TN8-19, QGHCTRWPWMCPPY (SEQ ID NO:33), and the linear peptides Linear-2 MEMLDSLFELLKDMVPISKA (SEQ ID NO:104); Linear-15 HHGWNYLRKGSAPQWFEAWV (SEQ. ID NO: 117); Linear-17RATLLKDFWQLVEGYGDN (SEQ ID NO: 119); Linear-20 YREMSMLEGLLDVLERLQHY (SEQID NO: 122), Linear-21 HNSSQMLLSELIMLVGSMMQ (SEQ ID NO: 123), Linear-24EFFHWLHNHRSEVNHWLDMN (SEQ ID NO: 126). Based on a consensus sequence,directed secondary phage display libraries were generated in which the“core” amino acids (determined from the consensus sequence) were eitherheld constant or biased in frequency of occurrence. Alternatively, anindividual peptide sequence could be used to generate a biased, directedphage display library. Panning of such libraries under more stringentconditions can yield peptides with enhanced binding to myostatin,selective binding to myostatin, or with some additional desiredproperty.

Production of Doped Oligos for Libraries

Oligonucleotides were synthesized in a DNA synthesizer which were 91%“doped” at the core sequences, that is, each solution was 91% of therepresented base (A, G, C, or T), and 3% of each of the other 3nucleotides. For the TN8-19 family, for example, a 91% doped oligo usedfor the construction of a secondary phage library was the following:

(SEQ ID NO: 634)5′-CAC AGT GCA CAG GGT NNK NNK NNK caK ggK caK tgK acK cgK tgKccK tgK atK tgK ccK ccK taK NNK NNK NNK CAT TCT CTC GAG ATC A-3′wherein “N” indicates that each of the four nucleotides A, T, C, and Gwere equally represented, K indicates that G and T were equallyrepresented, and the lower case letter represents a mixture of 91% ofthe indicated base and 3% of each of the other bases. The family ofoligonucleotides prepared in this manner were PCR amplified as describedabove, ligated into a phagemid vectors, for example, a modified pCES1plasmid (Dyax), or any available phagemid vector according to theprotocol described above. The secondary phage libraries generated wereall 91% doped and had between 1 and 6.5×10⁹ independent transformants.The libraries were panned as described above, but with the followingconditions:Round 1 Panning:

-   Input phage number: 10¹²-10¹³ cfu of phagemid-   Selection method: Nunc Immuno Tube selection-   Negative selection: 2× with Nunc Immuno Tubes coated with 2% BSA at    10 min each-   Panning coating: Coat with 1 μg of Myostatin protein in 1 ml of 0.1M    Sodium carbonate buffer (pH 9.6)-   Binding time: 3 hours-   Washing conditions: 6×2%-Milk-PBST; 6×PBST; 2×PBS-   Elution condition: 100 mM TEA elution    Round 2 Panning:-   Input phage number 10¹¹ cfu of phagemid-   Selection method: Nunc Immuno Tube selection-   Negative selection: 2× with Nunc Immuno Tubes coated with 2% BSA at    30 min. each-   Panning coating: Coat with 1 μg of Myostatin protein in 1 ml of 0.1M    Sodium carbonate buffer (pH 9.6)-   Binding time: 1 hour-   Washing conditions: 15×2%-Milk-PBST, 1×2%-Milk-PBST for 1 hr.,    10×2%-BSA-PBST, 1×2%-BSA-PBST for 1 hr., 10×PBST and 3×PBS-   Elution condition: 100 mM TEA elution    Round 3 Panning-   Input phage number: 10¹⁰ cfu of phagemid-   Selection method: Nunc Immuno Tube selection-   Negative selection: 6× with Nunc Immuno Tubes coated with 2% BSA at    10 min. each-   Panning coating: Coat with 0.1 μg of Myostatin protein in 1 ml of    0.1M Sodium carbonate buffer (pH 9.6)-   Binding time: 1 hour-   Washing conditions: 15×2%-Milk-PBST, 1×2%-Milk-PBST for 1 hr.,    10×2%-BSA-PBST, 1×2%-BSA-PBST for 1 hr., 10×PBST and 3×PBS-   Elution condition: 100 mM TEA elution

Panning of the secondary libraries yielded peptides with enhancedbinding to myostatin. Individual selected clones were subjected phageELISA, as described above, and sequenced.

The following affinity matured TN8-19 family of peptides are shown inTable IV below.

TABLE IV Affinity- SEQ matured ID peptibody NO: Peptide sequencemTN8-19-1 305 VALHGQCTRWPWMCPPQREG mTN8-19-2 306 YPEQGLCTRWPWMCPPQTLAmTN8-19-3 307 GLNQGHCTRWPWMCPPQDSN mTN8-19-4 308 MITQGQCTRWPWMCPPQPSGmTN8-19-5 309 AGAQEHCTRWPWMCAPNDWI mTN8-19-6 310 GVNQGQCTRWRWMCPPNGWEmTN8-19-7 311 LADHGQCIRWPWMCPPEGWE mTN8-19-8 312 ILEQAQCTRWPWMCPPQRGGmTN8-19-9 313 TQTHAQCTRWPWMCPPQWEG mTN8-19-10 314 VVTQGHCTLWPWMCPPQRWRmTN8-19-11 315 IYPHDQCTRWPWMCPPQPYP mTN8-19-12 316 SYWQGQCTRWPWMCPPQWRGmTN8-19-13 317 MWQQGHCTRWPWMCPPQGWG mTN8-19-14 318 EFTQWHCTRWPWMCPPQRSQmTN8-19-15 319 LDDQWQCTRWPWMCPPQGFS mTN8-19-16 320 YQTQGLCTRWPWMCPPQSQRmTN8-19-17 321 ESNQGQCTRWPWMCPPQGGW mTN8-19-18 322 WTDRGPCTRWPWMCPPQANGmTN8-19-19 323 VGTQGQCTRWPWMCPPYETG mTN8-19-20 324 PYEQGKCTRWPWMCPPYEVEmTN8-19-21 325 SEYQGLCTRWPWMCPPQGWK mTN8-19-22 326 TFSQGHCTRWPWMCPPQGWGmTN8-19-23 327 PGAHDHCTRWPWMCPPQSRY mTN8-19-24 328 VAEEWIICRRWPWMCPPQDWRmTN8-19-25 329 VGTQGHCTRWPWMCPPQPAG mTN8-19-26 330 EEDQAIICRSWPWMCPPQGWVmTN8-19-27 331 ADTQGHCTRWPWMCPPQHWF mTN8-19-28 332 SGPQGIICTRWPWMCAPQGWFmTN8-19-29 333 TLVQGHCTRWPWMCPPQRWV mTN8-19-30 334 GMAIIGKCTRWAWMCPPQSWKmTN8-19-31 335 ELYHGQCTRWPWMCPPQSWA mTN8-19-32 336VADIIGIICTRWPWMCPPQGWG mTN8-19-33 337 PESQGHCTRWPWMCPPQGWG mTN8-19-34338 IPAHGHCTRWPWMCPPQRWR mTN8-19-35 339 FTVHGHCTRWPWMCPPYGWV mTN8-19-36340 PDFPGHCTRWRWMCPPQGWE mTN8-19-37 341 QLWQGPCTQWPWMCPPKGRY mTN8-19-38342 HANDGHCTRWQWMCPPQWGG mTN8-19-39 343 ETDHGLCTRWPWMCPPYGAR mTN8-19-40344 GTWQGLCTRWPWMCPPQGWQ mTN8-19 con1 345 VATQGQCTRWPWMCPPQGWGmTN8-19 con2 346 VATQGQCTRWPWMCPPQRWG mTN8 con6-1 347QREWYPCYGGHLWCYDLHKA mTN8 con6-2 348 ISAWYSCYAGHFWCWDLKQK mTN8 con6-3349 WTGWYQCYGGHLWCYDLRRK mTN8 con6-4 350 KTFWYPCYDGHFWCYNLKSSmTN8 con6-5 351 ESRWYPCYEGHLWCFDLTET

The consensus sequence derived from the affinity-matured TN-8-19-1through Con2 (excluding the mTN8 con6 sequences) shown above is: Ca₁a₂Wa₃ WMCPP (SEQ ID NO: 352). All of these peptides comprise the sequenceWMCPP (SEQ ID NO: 633). The underlined amino acids represent the coreamino acids present in all embodiments, and a₁, a₂ and a₃ are selectedfrom a neutral hydrophobic, neutral polar, or basic amino acid. In oneembodiment of this consensus sequence, Cb₁b₂ Wb₃ WMCPP (SEQ ID NO: 353),b₁ is selected from any one of the amino acids T, I, or R; b₂ isselected from any one of R, S, Q; and b₃ is selected from any one of P,R and Q. All of the peptides comprise the sequence WMCPP (SEQ ID NO:633). A more detailed analysis of the affinity matured TN8 sequencescomprising SEQ ID NO: 352 provides the following formula:c₁c₂c₃c₄c₅c₆ Cc₇c₈ Wc₉ WMCPPc₁₀c₁₁c₁₂c₁₃

(SEQ ID NO: 354), wherein:

c₁ is absent or any amino acid;

c₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid; c₄ is absent or any amino acid;

c₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

c₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

c₇ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₈ is a neutral hydrophobic, neutral polar, or basic amino acid;

c₉ is a neutral hydrophobic, neutral polar or basic amino acid; andwherein

c₁₀ to C₁₃ is any amino acid.

In one embodiment of the above formulation, b₇ is selected from any oneof the amino acids T, I, or R; b₈ is selected from any one of R, S, Q;and b₉ is selected from any one of P, R and Q. This provides thefollowing sequence:d₁d₂d₃d₄d₅d₆ Cd₇d₈ Wd₉ WMCPPd₁₀d₁₁d₁₂d₁₃ (SEQ ID NO: 355).

d₁ is absent or any amino acid;

d₂ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₃ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₄ is absent or any amino acid;

d₅ is absent or a neutral hydrophobic, neutral polar, or acidic aminoacid;

d₆ is absent or a neutral hydrophobic, neutral polar, or basic aminoacid;

d₇ is selected from any one of the amino acids T, I, or R;

d₈ is selected from any one of R, S, Q;

d₉ is selected from any one of P, R and Q

and d₁₀ through d₁₃ are selected from any amino acid.

The consensus sequence of the mTN8 con6 series is WYc₁c₂ Yc₃ G, (SEQ IDNO: 356) wherein e₁ is P, S or Y; e₂ is C or Q, and e₃ is G or H.

In addition to the TN-19 affinity matured family, additional affinitymatured peptides were produced from the linear L-2, L-15, L-17, L-20,L-21, and L-24 first round peptides. These families are presented inTable V below.

TABLE V Affinity SEQ matured ID Peptide peptibody NO: Sequence L2 104MEMLDSLFELLKDMVPISKA mL2-Con1 357 RMEMLESLLELLKEIVPMSKAG mL2-Con2 358RMEMLESLLELLKEIVPMSKAR mL2-1 359 RMEMLESLLELLKDIVPMSKPS mL2-2 360GMEMLESLFELLQEIVPMSKAP mL2-3 361 RMEMLESLLELLKDIVPISNPP mL2-4 362RIEMLESLLELLQEIVPISKAE mL2-5 363 RMEMLQSLLELLKDIVPMSNAR mL2-6 364RMEMLESLLELLKEIVPTSNGT mL2-7 365 RMEMLESLFELLKEIVPMSKAG mL2-8 366RMEMLGSLLELLKEIVPMSKAR mL2-9 367 QMELLDSLFELLKEIVPKSQPA mL2-10 368RMEMLDSLLELLKEIVPMSNAR mL2-11 369 RMEMLESLLELLHEIVPMSQAG mL2-12 370QMEMLESLLQLLKEIVPMSKAS mL2-13 371 RMEMLDSLLELLKDMVPMTTGA mL2-14 372RIEMLESLLELLKDMVPMANAS mL2-15 373 RMEMLESLLQLLNEIVPMSRAR mL2-16 374RMEMLESLFDLLKELVPMSKGV mL2-17 375 RIEMLESLLELLKDIVPIQKAR mL2-18 376RMELLESLFELLKDMVPMSDSS mL2-19 377 RMEMLESLLEVLQEIVPRAKGA mL2-20 378RMEMLDSLLQLLNEIVPMSHAR mL2-21 379 RMEMLESLLELLKDIVPMSNAG mL2-22 380RMEMLQSLFELLKGMVPISKAG mL2-23 381 RMEMLESLLELLKEIVPNSTAA mL2-24 382RMEMLQSLLELLKEIVPISKAG mL2-25 383 RIEMLDSLLELLNELVPMSKAR L-15 117HHGWNYLRKGSAPQWFEAWV mL15-con1 384 QVESLQQLLMWLDQKLASGPQG mL15-1 385RMELLESLFELLKEMVPRSKAV mL15-2 386 QAVSLQHLLMWLDQKLASGPQH mL15-3 387DEDSLQQLLMWLDQKLASGPQL mL15-4 388 PVASLQQLLIWLDQKLAQGPHA mL15-5 389EVDELQQLLNWLDHKLASGPLQ mL15-6 390 DVESLEQLLMWLDHQLASGPHG mL15-7 391QVDSLQQVLLWLEHKLALGPQV mL15-8 392 GDESLQHLLMWLLQKLALGPHG mL15-9 393QIEMLESLLDLLRDMVPMSNAF mL15-10 394 EVDSLQQLLMWLDQKLASGPQA mL15-11 395EDESLQQLLIYLDKMLSSGPQV mL15-12 396 AMDQLHQLLIWLDHKLASGPQA mL15-13 397RIEMLESLLELLDEIALIPKAW mL15-14 398 EVVSLQHLLMWLEHKLASGPDG mL15-15 399GGESLQQLLMWLDQQLASGPQR mL15-16 400 GVESLQQLLIFLDHMLVSGPHD mL15-17 401NVESLEHLMMWLERLLASGPYA mL15-18 402 QVDSLQQLLIWLDHQLASGPKR mL15-19 403EVESLQQLLMWLEHKLAQGPQG mL15-20 404 EVDSLQQLLMWLDQKLASGPHA mL15-21 405EVDSLQQLLMWLDQQLASGPQK mL15-22 406 GVEQLPQLLMWLEQKLASGPQR mL15-23 407GEDSLQQLLMWLDQQLAAGPQV mL15-24 408 ADDSLQQLLMWLDRKLASGPHV mL15-25 409PVDSLQQLLIWLDQKLASGPQG L-17 119 RATLLKDFWQLVEGYGDN mL17-con1 410DWRATLLKEFWQLVEGLGDNLV mL17-con2 411 QSRATLLKEFWQLVEGLGDKQA m117-1 412DGRATLLTEFWQLVQGLGQKEA mL17-2 413 LARATLLKEFWQLVEGLGEKVV mL17-3 414GSRDTLLKEFWQLVVGLGDMQT mL17-4 415 DARATLLKEFWQLVDAYGDRMV mL17-5 416NDRAQLLRDFWQLVDGLGVKSW mL17-6 417 GVRETLLYELWYLLKGLGANQG mL17-7 418QARATLLKEFCQLVGCQGDKLS mL17-8 419 QERATLLKEFWQLVAGLGQNMR mL17-9 420SGRATLLKEFWQLVQGLGEYRW mL17-10 421 TMRATLLKEFWLFVDGQREMQW mL17-11 422GERATLLNDFWQLVDGQGDNTG mL17-12 423 DERETLLKEFWQLVHGWGDNVA mL17-13 424GGRATLLKELWQLLEGQGANLV mL17-14 425 TARATLLNELVQLVKGYGDKLV mL17-15 426GMRATLLQEFWQLVGGQGDNWM mL17-16 427 STRATLLNDLWQLMKGWAEDRG mL17-17 428SERATLLKELWQLVGGWGDNFG mL17-18 429 VGRATLLKEFWQLVEGLVGQSR mL17-19 430EIRATLLKEFWQLVDEWREQPN mL17-20 431 QLRATLLKEFLQLVHGLGETDS mL17-21 432TQRATLLKEFWQLIEGLGGKHV mL17-22 433 HYRATLLKEFWQLVDGLREQGV mL17-23 434QSRVTLLREFWQLVESYRPIVN mL17-24 435 LSRATLLNEFWQFVDGQRDKRM mL17-25 436WDRATLLNDFWHLMEELSQKPG mL17-26 437 QERATLLKEFWRMVEGLGKNRG mL17-27 438NERATLLREFWQLVGGYGVNQR L-20 122 YREMSMLEGLLDVLERLQHY mL20-1 439HQRDMSMLWELLDVLDGLRQYS mL20-2 440 TQRDMSMLDGLLEVLDQLRQQR mL20-3 441TSRDMSLLWELLEELDRLGHQR mL20-4 442 MQIIDMSMLYGLVELLESLGIIQI mL20-5 443WNRDMRMLESLFEVLDGLRQQV mL20-6 444 GYRDMSMLEGLLAVLDRLGPQL mL20 con1 445TQRDMSMLEGLLEVLDRLGQQR mL20 con2 446 WYRDMSMLEGLLEVLDRLGQQR L-21 123HNSSQMLLSELIMLVGSMMQ mL21-1 447 TQNSRQMLLSDFMMLVGSMIQG mL21-2 448MQTSRHILLSEFMMLVGSIMHG mL21-3 449 HDNSRQMLLSDLLHLVGTMIQG mL21-4 450MENSRQNLLRELIMLVGNMSHQ mL21-5 451 QDTSRHMLLREFMMLVGEMIQG mL21 con1 452DQNSRQMLLSDLMILVGSMIQG L-24 126 EFFHWLHNHRSEVNHWLDMN mL24-1 453NVFFQWVQKHGRVVYQWLDINV mL24-2 454 FDFLQWLQNHRSEVEHWLVMDV

The affinity matured peptides provided in Tables IV and V are thenassembed into peptibodies as described above and assayed using the invivo assays.

The affinity matured L² peptides comprise a consensus sequence of f₁EMLf ₂ SLf₃f₄ LL, (SEQ ID NO: 455), wherein f₁ is M or I; f₂ is anyamino acid; f₃ is L or F; and f₄ is E, Q or D.

The affinity matured L¹⁵ peptide family comprise the sequence Lg₁g₂LLg₃g₄ L, (SEQ ID NO: 456), wherein g₁ is Q, D or E, g₂ is S, Q, D or E,g₃ is any amino acid, and g₄ is L, W, F, or Y. The affinity matured L¹⁷family comprises the sequence: h₁h₂h₃h₄h₅h₆h₇h₈h₉ (SEQ ID NO: 457)wherein h₁ is R or D; h₂ is any amino acid; h₃ is A, T S or Q; h₄ is Lor M; h₅ is L or S; h₆ is any amino acid; h₇ is F or E; h₈ is W, F or C;and h₉ is L, F, M or K. Consensus sequences may also be determined forthe mL20, mL21 and mL24 families of peptides shown above.

Peptibodies were constructed from these affinity matured peptides asdescribed above, using a linker attached to the Fc domain of human IgG1,having SEQ ID NO: 296, at the N-terminus (N configuration), at the Cterminus (C configuration) of the Fe, or at both the N and C terminals(N,C configurations), as described in Example 2 above. The peptidesnamed were attached to the C or N terminals via a 5 glycine (5G), 8glycine or k linker sequence. In the 2× peptibody version the peptideswere linked with linkers such as 5 gly, 8 gly or k. Affinity maturedpeptides and peptibodies are designated with a small “m” such asmTN8-19-22 for example. Peptibodies of the present invention furthercontain two splice sites where the peptides were spliced into thephagemid vectors. The position of these splice sites are AQ-peptide-LE.The peptibodies generally include these additional amino acids (althoughthey are not included in the peptide sequences listed in the tables). Insome peptibodies the LE amino acids were removed from the peptidessequences. These peptibodies are designated -LE.

Exemplary peptibodies, and exemplary polynucleotide sequences encodingthem, are provided in Table VI below. This table includes examples ofpeptibody sequences (as opposed to peptide only), such as the 2×mTN8-19-7 (SEQ ID NO: 615) and the peptibody with the LE sequencesdeleted (SEQ ID NO: 617). By way of explanation, the linker sequences inthe 2× versions refer to the linker between the tandem peptides. Thesepeptibody sequences contain the Fc, linkers, AQ and LE sequences. Theaccompanying nucleotide sequence encodes the peptide sequence inaddition to the AQ/LE linker sequences, if present, but does not encodethe designated linker.

TABLE VI Peptibody Nucleotide Sequence Name Peptide (SEQ ID No) LinkerTerminus mL2-Con1 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKEIVPMSKAG TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 357) AATGTCTAAAGCTGGT(SEQ ID NO: 458) mL2-Con2 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKEIVPMSKAR TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 358) AATGTCTAAAGCTCGT(SEQ ID NO: 459) mL2-1 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKDIVPMSKPS TTGAACTTCTTAAAGATATTGTTCC (SEQ ID NO: 359) AATGTCTAAACCATCT(SEQ ID NO: 460) mL2-2 GMEMLESLFELL GGTATGGAAATGCTTGAATCTCTTT 5 gly NQEIVPMSKAP TTGAACTTCTTCAAGAAATTGTTCC (SEQ ID NO: 360) AATGTCTAAAGCTCCA(SEQ ID NO: 461) mL2-3 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKDIVPISNPP TTGAACTTCTTAAAGATATTGTTCC (SEQ ID NO: 361) AATTTCTAATCCACCA(SEQ ID NO: 462) mL2-4 RIEMLESLLELLQ CGTATTGAAATGCTTGAATCTCTTC 5 gly NEIVPISKAE TTGAACTTCTTCAAGAAATTGTTCC (SEQ ID NO: 362) AATTTCTAAAGCTGAA(SEQ ID NO: 463) mL2-5 RMEMLQSLLELL CGTATGGAAATGCTTCAATCTCTTC 5 gly NKDIVPMSNAR TTGAACTTCTTAAAGATATTGTTCC (SEQ ID NO: 363) AATGTCTAATGCTCGT(SEQ ID NO: 464) mL2-6 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKEIVPTSNGT TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 364) AACTTCTAATGGTACT(SEQ ID NO: 465) mL2-7 RMEMLESLFELL CGTATGGAAATGCTTGAATCTCTTT 5 gly NKEIVPMSKAG TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 365) AATGTCTAAAGCTGGT(SEQ ID NO: 466) mL2-8 RMEMLGSLLELL CGTATGGAAATGCTTGGTTCTCTTC 5 gly NKEIVPMSKAR TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 366) AATGTCTAAAGCTCGT(SEQ ID NO: 467) mL2-9 QMELLDSLFELL CAAATGGAACTTCTTGATTCTCTTT 5 gly NKEIVPKSQPA TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 367) AAAATCTCAACCAGCT(SEQ ID NO: 468) mL2-10 RMEMLDSLLELL CGTATGGAAATGCTTGATTCTCTTC 5 gly NKEIVPMSNAR TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 368) AATGTCTAATGCTCGT(SEQ ID NO: 469) mL2-11 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NHEIVPMSQAG TTGAACTTCTTCATGAAATTGTTCC (SEQ ID NO: 369) AATGTCTCAAGCTGGT(SEQ ID NO: 470) mL2 12 QMEMLESLLQLL CAAATGGAAATGCTTGAATCTCTTC 5 gly NKEIVPMSKAS TTCAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 370) AATGTCTAAAGCTTCT(SEQ ID NO: 471) mL2-13 RMEMLDSLLELL CGTATGGAAATGCTTGATTCTCTTC 5 gly NKDMVPMTTGA TTGAACTTCTTAAAGATATGGTTCC (SEQ ID NO: 371) AATGACTACTGGTGCT(SEQ ID NO: 472) mL2-14 RIEMLESLLELLK CGTATTGAAATGCTTGAATCTCTTC 5 gly NDMVPMANAS TTGAACTTCTTAAAGATATGGTTCC (SEQ ID NO: 372) AATGGCTAATGCTTCT(SEQ ID NO: 473) mL2-15 RMEMLESLLQLL CGTATGGAAATGCTTGAATCTCTTC 5 gly NNEIVPMSRAR TTCAACTTCTTAATGAAATTGTTCC (SEQ ID NO: 373) AATGTCTCGTGCTCGT(SEQ ID NO: 474) mL2-16 RMEMLESLFDLL CGTATGGAAATGCTTGAATCTCTTT 5 gly NKELVPMSKGV TTGATCTTCTTAAAGAACTTGTTCC (SEQ ID NO: 374) AATGTCTAAAGGTGTT(SEQ ID NO: 475) mL2-17 RIEMLESLLELLK CGTATTGAAATGCTTGAATCTCTTC 5 gly NDIVPIQKAR TTGAACTTCTTAAAGATATTGTTCC (SEQ ID NO: 375) AATTCAAAAAGCTCGT(SEQ ID NO: 476) mL2-18 RMELLESLFELLK CGTATGGAACTTCTTGAATCTCTTT 5 gly NDMVPMSDSS TTGAACTTCTTAAAGATATGGTTCC (SEQ ID NO: 376) AATGTCTGATTCTTCT(SEQ ID NO: 477) mL2-19 RMEMLESLLEVL CGTATGGAAATGCTTGAATCTCTTC 5 gly NQEIVPRAKGA TTGAAGTTCTTCAAGAAATTGTTCC (SEQ ID NO: 377) ACGTGCTAAAGGTGCT(SEQ ID NO: 478) mL2-20 RMEMLDSLLQLL CGTATGGAAATGCTTGATTCTCTTC 5 gly NNEIVPMSHAR TTCAACTTCTTAATGAAATTGTTCC (SEQ ID NO: 378) AATGTCTCATGCTCGT(SEQ ID NO: 479) mL2-21 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKDIVPMSNAG TTGAACTTCTTAAAGATATTGTTCC (SEQ ID NO: 379) AATGTCTAATGCTGGT(SEQ ID NO: 480) mL2-22 RMEMLQSLFELL CGTATGGAAATGCTTCAATCTCTTT 5 gly NKGMVPISKAG TTGAACTTCTTAAAGGTATGGTTCC (SEQ ID NO: 380) AATTTCTAAAGCTGGT(SEQ ID NO: 481) mL2-23 RMEMLESLLELL CGTATGGAAATGCTTGAATCTCTTC 5 gly NKEIVPNSTAA TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 381) AAATTCTACTGCTGCT(SEQ ID NO: 482) mL2-24 RMEMLQSLLELL CGTATGGAAATGCTTCAATCTCTTC 5 gly NKEIVPISKAG TTGAACTTCTTAAAGAAATTGTTCC (SEQ ID NO: 382) AATTTCTAAAGCTGGT(SEQ ID NO: 483) mL2-25 RIEMLDSLLELLN CGTATTGAAATGCTTGATTCTCTTC 5 gly NELVPMSKAR TTGAACTTCTTAATGAACTTGTTCC (SEQ ID NO: 383) AATGTCTAAAGCTCGT(SEQ ID NO: 484) mL17 Con1 DWRATLLKEFW GATTGGCGTGCTACTCTTCTTAAAG 5 gly NQLVEGLGDNLV AATTTTGGCAACTTGTTGAAGGTCT (SEQ ID NO: 384) TGGTGATAATCTTGTT(SEQ ID NO: 485) mL17-1 DGRATLLTEFWQ GATGGTCGTGCTACTCTTCTTACTG 5 gly NLVQGLGQKEA AATTTTGGCAACTTGTTCAAGGTCT (SEQ ID NO: 412) TGGTCAAAAAGAAGCT(SEQ ID NO: 486) mL17-2 LARATLLKEFWQ CTTGCTCGTGCTACTCTTCTTAAAG 5 gly NLVEGLGEKVV AATTTTGGCAACTTGTTGAAGGTCT (SEQ ID NO: 413) TGGTGAAAAAGTTGTT(SEQ ID NO: 487) mL17-3 GSRDTLLKEFWQ GGTTCTCGTGATACTCTTCTTAAAG 5 gly NLVVGLGDMQT AATTTTGGCAACTTGTTGTTGGTCT (SEQ ID NO: 414) TGGTGATATGCAAACT(SEQ ID NO: 488) mL17-4 DARATLLKEFWQ GATGCTCGTGCTACTCTTCTTAAAG 5 gly NLVDAYGDRMV AATTTTGGCAACTTGTTGATGCTTA (SEQ ID NO: 415) TGGTGATCGTATGGTT(SEQ ID NO: 489) mL17-5 NDRAQLLRDFWQ AATGATCGTGCTCAACTTCTTCGTG 5 gly NLVDGLGVKSW ATTTTTGGCAACTTGTTGATGGTCT (SEQ ID NO: 416) TGGTGTTAAATCTTGG(SEQ ID NO: 490) mL17-6 GVRETLLYELWY GGTGTTCGTGAAACTCTTCTTTATG 5 gly NLLKGLGANQG AACTTTGGTATCTTCTTAAAGGTCT (SEQ ID NO: 417) TGGTGCTAATCAAGGT(SEQ ID NO: 491) mL17-7 QARATLLKEFCQ CAAGCTCGTGCTACTCTTCTTAAAG 5 gly NLVGCQGDKLS AATTTTGTCAACTTGTTGGTTGTCA (SEQ ID NO: 418) AGGTGATAAACTTTCT(SEQ ID NO: 492) mL17-8 QERATLLKEFWQ CAAGAACGTGCTACTCTTCTTAAA 5 gly NLVAGLGQNMR GAATTTTGGCAACTTGTTGCTGGTC (SEQ ID NO: 419) TTGGTCAAAATATGCGT(SEQ ID NO: 493) mL17-9 SGRATLLKEFWQ TCTGGTCGTGCTACTCTTCTTAAAG 5 gly NLVQGLGEYRW AATTTTGGCAACTTGTTCAAGGTCT (SEQ ID NO: 420) TGGTGAATATCGTTGG(SEQ ID NO: 494) mL17-10 TMRATLLKEFWL ACTATGCGTGCTACTCTTCTTAAAG 5 gly NFVDGQREMQW AATTTTGGCTTTTTGTTGATGGTCA (SEQ ID NO: 421) ACGTGAAATGCAATGG(SEQ ID NO: 495) mL17-11 GERATLLNDFWQ GGTGAACGTGCTACTCTTCTTAATG 5 gly NLVDGQGDNTG ATTTTTGGCAACTTGTTGATGGTCA (SEQ ID NO: 422) AGGTGATAATACTGGT(SEQ ID NO: 496) mL17-12 DERETLLKEFWQ GATGAACGTGAAACTCTTCTTAAA 5 gly NLVHGWGDNVA GAATTTTGGCAACTTGTTCATGGTT (SEQ ID NO: 423) GGGGTGATAATGTTGCT(SEQ ID NO: 497) mL17-13 GGRATLLKELWQ GGTGGTCGTGCTACTCTTCTTAAAG 5 gly NLLEGQGANLV AACTTTGGCAACTTCTTGAAGGTCA (SEQ ID NO: 424) AGGTGCTAATCTTGTT(SEQ ID NO: 498) mL17 14 TARATLLNELVQ ACTGCTCGTGCTACTCTTCTTAATG 5 gly NLVKGYGDKLV AACTTGTTCAACTTGTTAAAGGTTA (SEQ ID NO: 425) TGGTGATAAACTTGTT(SEQ ID NO: 499) mL17-15 GMRATLLQEFWQ GGTATGCGTGCTACTCTTCTTCAAG 5 gly NLVGGQGDNWM AATTTTGGCAACTTGTTGGTGCTCA (SEQ ID NO: 426) AGGTGATAATTGGATG(SEQ ID NO: 500) mL17-16 STRATLLNDLWQ TCTACTCGTGCTACTCTTCTTAATG 5 gly NLMKGWAEDRG ATCTTTGGCAACTTATGAAAGGTTG (SEQ ID NO: 427) GGCTGAAGATCGTGGT(SEQ ID NO: 501) mL17-17 SERATLLKELWQ TCTGAACGTGCTACTCTTCTTAAAG 5 gly NLVGGWGDNFG AACTTTGGCAACTTGTTGGTGGTTG (SEQ ID NO: 428) GGGTGATAATTTTGGT(SEQ ID NO: 502) mL17-18 VGRATLLKEFWQ GTTGGTCGTGCTACTCTTCTTAAAG 5 gly NLVEGLVGQSR AATTTTGGCAACTTGTTGAAGGTCT (SEQ ID NO: 429) TGTTGGTCAATCTCGT(SEQ ID NO: 503) 2x mTN8-Con6- M-GAQ- TGGTATCCGTGTTATGAGGGTCACT 1K N(N)-1K WYPCYEGHFWC TCTGGTGCTACGATCTGGGTTCTGG YDL-TTCCACTGCTTCTTCTGGTTCCGGT GSGSATGGSGST TCCGCTACTGGTTGGTACCCGTGCTASSGSGSATG- ACGAAGGTCACTTTTGGTGTTATGA WYPCYEGHFWC TCTG YDL-LE-5G-FC(SEQ ID NO: 505) (SEQ ID NO: 504) 2x mTN8-Con6- FC-5G-AQ-TGGTATCCGTGTTATGAGGGTCACT 1K C (C)-1K WYPCYEGIIFWCTCTGGTGCTACGATCTGGGTTCTGG YDL- TTCCACTGCTTCTTCTGGTTCCGGT GSGSATGGSGSTTCCGCTACTGGTTGGTACCCGTGCT ASSGSGSATG- ACGAAGGTCACTTTTGGTGTTATGAWYPCYEGHFWC TCTG YDL-LE (SEQ ID NO: 507) (SEQ ID NO: 506) 2x mTN8-Con7-M-GAQ- ATCTTTGGCTGTAAATGGTGGGAC 1K N (N)-1K IFGCKWWDVQCGTTCAGTGCTACCAGTTCGGTTCTG YQF- GTTCCACTGCTTCTTCTGGTTCCGG GSGSATGGSGSTTTCCGCTACTGGTATCTTCGGTTGC ASSGSGSATG- AAGTGGTGGGATGTACAGTGTTATIFGCKWWDVQC CAGTTT YQF-LE-5G-FC (SEQ ID NO: 509) (SEQ ID NO: 508)2x mTN8-Con7- FC-5G-AQ- ATCTTTGGCTGTAAATGGTGGGAC 1K C (C) 1K IFGCKWWDVQCGTTCAGTGCTACCAGTTCGGTTCTG YQF- GTTCCACTGCTTCTTCTGGTTCCGG GSGSATGGSGSTTTCCGCTACTGGTATCTTCGGTTGC ASSGSGSATG- AAGTGGTGGGATGTACAGTGTTATIFGCKWWDVQC CAGTTT YQF-LE (SEQ ID NO: 511) (SEQ ID NO: 510)2x mTN8-Con8- M-GAQ- ATCTTTGGCTGTAAGTGGTGGGAC 1K N (N)-1K IFGCKWWDVDCGTTGACTGCTACCAGTTCGGTTCTG YQF- GTTCCACTGCTTCTTCTGGTTCCGG GSGSATGGSGSTTTCCGCTACTGGTATCTTCGGTTGC ASSGSGSATG- AAATGGTGGGACGTTGATTGTTATIFGCKWWDVDC CAGTTT YQF LE 5G FC (SEQ ID NO: 513) (SEQ ID NO: 512)2x mTN8-Con8- FC-5G-AQ- ATCTTTGGCTGTAAGTGGTGGGAC 1K C (C)-1K IFGCKWWDVDCGTTGACTGCTACCAGTTCGGTTCTG YQF- GTTCCACTGCTTCTTCTGGTTCCGG GSGSATGGSGSTTTCCGCTACTGGTATCTTCGGTTGC ASSGSGSATG- AAATGGTGGGACGTTGATTGTTATIFGCKWWDVDC CAGTTT YQF-LE (SEQ ID NO: 515) (SEQ ID NO: 514) mL15-Con1QVESLQQLLMWL CAGGTTGAATCCCTGCAGCAGCTG 5 gly C DQKLASGPQGCTGATGTGGCTGGACCAGAAACTG (SEQ ID NO: 384) GCTTCCGGTCCGCAGGGT(SEQ ID NO: 516) mL15-1 RMELLESLFELLK CGTATGGAACTGCTGGAATCCCTG 5 gly CEMVPRSKAV TTCGAACTGCTGAAAGAAATGGTT (SEQ ID NO: 385) CCGCGTTCCAAAGCTGTT(SEQ ID NO: 517) mL15-2 QAVSLQHLLMW CAGGCTGTTTCCCTGCAGCACCTGC 5 gly CLDQKLASGPQH TGATGTGGCTGGACCAGAAACTGG (SEQ ID NO: 386) CTTCCGGTCCGCAGCAC(SEQ ID NO: 518) mL15-3 DEDSLQQLLMWL GACGAAGACTCCCTGCAGCAGCTG 5 gly CDQKLASGPQL CTGATGTGGCTGGACCAGAAACTG (SEQ ID NO: 387) GCTTCCGGTCCGCAGCTG(SEQ ID NO: 519) mL15-4 PVASLQQLLIWL CCGGTTGCTTCCCTGCAGCAGCTGC 5 gly CDQKLAQGPHA TGATCTGGCTGGACCAGAAACTGG (SEQ ID NO: 388) CTCAGGGTCCGCACGCT(SEQ ID NO: 520) mL15-5 EVDELQQLLNWL GAAGTTGACGAACTGCAGCAGCTG 5 gly CDHKLASGPLQ CTGAACTGGCTGGACCACAAACTG (SEQ ID NO: 389) GCTTCCGGTCCGCTGCAG(SEQ ID NO: 521) mL15-6 DVESLEQLLMWL GACGTTGAATCCCTGGAACAGCTG 5 gly CDHQLASGPHG CTGATGTGGCTGGACCACCAGCTG (SEQ ID NO: 390) GCTTCCGGTCCGCACGGT(SEQ ID NO: 522) mL15-7 QVDSLQQVLLWL CAGGTTGACTCCCTGCAGCAGGTT 5 gly CEIIKLALGPQV CTGCTGTGGCTGGAACACAAACTG (SEQ ID NO: 391) GCTCTGGGTCCGCAGGTT(SEQ ID NO: 523) mL15 8 GDESLQHLLMWL GGTGACGAATCCCTGCAGCACCTG 5 gly CEQKLALGPHG CTGATGTGGCTGGAACAGAAACTG (SEQ ID NO: 392) GCTCTGGGTCCGCACGGT(SEQ ID NO: 524) mL15-9 QIEMLESLLDLLR CAGATCGAAATGCTGGAATCCCTG 5 gly CDMVPMSNAF CTGGACCTGCTGCGTGACATGGTTC (SEQ ID NO: 393) CGATGTCCAACGCTTTC(SEQ ID NO: 525) mL15-10 EVDSLQQLLMWL GAAGTTGACTCCCTGCAGCAGCTG 5 gly CDQKLASGPQA CTGATGTGGCTGGACCAGAAACTG (SEQ ID NO: 394) GCTTCCGGTCCGCAGGCT(SEQ ID NO: 526) mL15-11 EDESLQQLLIYLD GAAGACGAATCCCTGCAGCAGCTG 5 gly CKMLSSGPQV CTGATCTACCTGGACAAAATGCTG (SEQ ID NO: 395) TCCTCCGGTCCGCAGGTT(SEQ ID NO: 527) mL15-12 AMDQLHQLLIWL GCTATGGACCAGCTGCACCAGCTG 5 gly CDHKLASGPQA CTGATCTGGCTGGACCACAAACTG (SEQ ID NO: 396) GCTTCCGGTCCGCAGGCT(SEQ ID NO: 528) mL15-13 RIEMLESLLELLD CGTATCGAAATGCTGGAATCCCTG 5 gly CEIALIPKAW CTGGAACTGCTGGACGAAATCGCT (SEQ ID NO: 397) CTGATCCCGAAAGCTTGG(SEQ ID NO: 529) mL15-14 EVVSLQHLLMWL GAAGTTGTTTCCCTGCAGCACCTGC 5 gly CEHKLASGPDG TGATGTGGCTGGAACACAAACTGG (SEQ ID NO: 398) CTTCCGGTCCGGACGGT(SEQ ID NO: 530) mL15-15 GGESLQQLLMWL GGTGGTGAATCCCTGCAGCAGCTG 5 gly CDQQLASGPQR CTGATGTGGCTGGACCAGCAGCTG (SEQ ID NO: 399) GCTTCCGGTCCGCAGCGT(SEQ ID NO: 531) mL15-16 GVESLQQLLIFLD GGTGTTGAATCCCTGCAGCAGCTG 5 gly CHMLVSGPHD CTGATCTTCCTGGACCACATGCTGG (SEQ ID NO: 400) TTTCCGGTCCGCACGAC(SEQ ID NO: 532) mL15-17 NVESLEHLMMW AACGTTGAATCCCTGGAACACCTG 5 gly CLERLLASGPYA ATGATGTGGCTGGAACGTCTGCTG (SEQ ID NO: 401) GCTTCCGGTCCGTACGCT(SEQ ID NO: 533) mL15-18 QVDSLQQLLIWL CAGGTTGACTCCCTGCAGCAGCTG 5 gly CDHQLASGPKR CTGATCTGGCTGGACCACCAGCTG (SEQ ID NO: 402) GCTTCCGGTCCGAAACGT(SEQ ID NO: 534) mL15-19 EVESLQQLLMWL GAAGTTGAATCCCTGCAGCAGCTG 5 gly CEHKLAQGPQG CTGATGTGGCTGGAACACAAACTG (SEQ ID NO: 403) GCTCAGGGTCCGCAGGGT(SEQ ID NO: 535) mL15-20 EVDSLQQLLMWL GAAGTTGACTCCCTGCAGCAGCTG 5 gly CDQKLASGPHA CTGATGTGGCTGGACCAGAAACTG (SEQ ID NO: 404) GCTTCCGGTCCGCACGCT(SEQ ID NO: 536) mL15-21 EVDSLQQLLMWL GAAGTTGACTCCCTGCAGCAGCTG 5 gly CDQQLASGPQK CTGATGTGGCTGGACCAGCAGCTG (SEQ ID NO: 405) GCTTCCGGTCCGCAGAAA(SEQ ID NO: 537) mL15 22 GVEQLPQLLMWL GGTGTTGAACAGCTGCCGCAGCTG 5 gly CEQKLASGPQR CTGATGTGGCTGGAACAGAAACTG (SEQ ID NO: 406) GCTTCCGGTCCGCAGCGT(SEQ ID NO: 538) mL15-23 GEDSLQQLLMWL GGTGAAGACTCCCTGCAGCAGCTG 5 gly CDQQLAAGPQV CTGATGTGGCTGGACCAGCAGCTG (SEQ ID NO: 407) GCTGCTGGTCCGCAGGTT(SEQ ID NO: 539) mL15-24 ADDSLQQLLMW GCTGACGACTCCCTGCAGCAGCTG 5 gly CLDRKLASGPHV CTGATGTGGCTGGACCGTAAACTG (SEQ ID NO: 408) GCTTCCGGTCCGCACGTT(SEQ ID NO: 540) mL15-25 PVDSLQQLLIWL CCGGTTGACTCCCTGCAGCAGCTG 5 gly CDQKLASGPQG CTGATCTGGCTGGACCAGAAACTG (SEQ ID NO: 409) GCTTCCGGTCCGCAGGGT(SEQ ID NO: 541) mL17-con2 QSRATLLKEFWQ CAGTCCCGTGCTACCCTGCTGAAA 5 gly CLVEGLGDKQA GAATTCTGGCAGCTGGTTGAAGGT (SEQ ID NO: 411) CTGGGTGACAAACAGGCT(SEQ ID NO: 542) mL17-19 EIRATLLKEFWQL GAAATCCGTGCTACCCTGCTGAAA 5 gly CVDEWREQPN GAATTCTGGCAGCTGGTTGACGAA (SEQ ID NO: 430) TGGCGTGAACAGCCGAAC(SEQ ID NO: 543) mL17-20 QLRATLLKEFLQL CAGCTGCGTGCTACCCTGCTGAAA 5 gly CVHGLGETDS GAATTCCTGCAGCTGGTTCACGGTC (SEQ ID NO: 431) TGGGTGAAACCGACTCC(SEQ ID NO: 544) mL17-21 TQRATLLKEFWQ ACCCAGCGTGCTACCCTGCTGAAA 5 gly CLIEGLGGKHV GAATTCTGGCAGCTGATCGAAGGT (SEQ ID NO: 432) CTGGGTGGTAAACACGTT(SEQ ID NO: 545) mL17-22 HYRATLLKEFWQ CACTACCGTGCTACCCTGCTGAAA 5 gly CLVDGLREQGV GAATTCTGGCAGCTGGTTGACGGT (SEQ ID NO: 433) CTGCGTGAACAGGGTGTT(SEQ ID NO: 546) mL17-23 QSRVTLLREFWQ CAGTCCCGTGTTACCCTGCTGCGTG 5 gly CLVESYRPIVN AATTCTGGCAGCTGGTTGAATCCTA (SEQ ID NO: 434) CCGTCCGATCGTTAAC(SEQ ID NO: 547) mL17-24 LSRATLLNEFWQ CTGTCCCGTGCTACCCTGCTGAACG 5 gly CFVDGQRDKRM AATTCTGGCAGTTCGTTGACGGTCA (SEQ ID NO: 435) GCGTGACAAACGTATG(SEQ ID NO: 548) mL17-25 WDRATLLNDFW TGGGACCGTGCTACCCTGCTGAAC 5 gly CHLMEELSQKPG GACTTCTGGCACCTGATGGAAGAA (SEQ ID NO: 436) CTGTCCCAGAAACCGGGT(SEQ ID NO: 549) mL17-26 QERATLLKEFWR CAGGAACGTGCTACCCTGCTGAAA 5 gly CMVEGLGKNRG GAATTCTGGCGTATGGTTGAAGGT (SEQ ID NO: 437) CTGGGTAAAAACCGTGGT(SEQ ID NO: 550) mL17-27 NERATLLREFWQ AACGAACGTGCTACCCTGCTGCGT 5 gly CLVGGYGVNQR GAATTCTGGCAGCTGGTTGGTGGTT (SEQ ID NO: 438) ACGGTGTTAACCAGCGT(SEQ ID NO: 551) mTN8Con6 1 QREWYPCYGGHL CAGCGTGAATGGTACCCGTGCTAC 5 glyC WCYDLHKA GGTGGTCACCTGTGGTGCTACGAC (SEQ ID NO: 347) CTGCACAAAGCT(SEQ ID NO: 552) mTN8Con6-2 ISAWYSCYAGHF ATCTCCGCTTGGTACTCCTGCTACG 5 glyC WCWDLKQK CTGGTCACTTCTGGTGCTGGGACCT (SEQ ID NO: 348) GAAACAGAAA(SEQ ID NO: 553) mTN8Con6-3 WTGWYQCYGGH TGGACCGGTTGGTACCAGTGCTAC 5 gly CLWCYDLRRK GGTGGTCACCTGTGGTGCTACGAC (SEQ ID NO: 349) CTGCGTCGTAAA(SEQ ID NO: 554) mTN8Con6 4 KTFWYPCYDGHF AAAACCTTCTGGTACCCGTGCTAC 5 glyC WCYNLKSS GACGGTCACTTCTGGTGCTACAAC (SEQ ID NO: 350) CTGAAATCCTCC(SEQ ID NO: 555) mTN8Con6-5 ESRWYPCYEGHL GAATCCCGTTGGTACCCGTGCTAC 5 glyC WCFDLTET GAAGGTCACCTGTGGTGCTTCGAC (SEQ ID NO: 351) CTGACCGAAACC(SEQ ID NO: 556) mL24-1 NVFFQWVQKHG AATGTTTTTTTTCAATGGGTTCAAA 5 gly CRVVYQWLDINV AACATGGTCGTGTTGTTTATCAATG (SEQ ID NO: 453) GCTTGATATTAATGTT(SEQ ID NO: 557) mL24-2 FDFLQWLQNHRS TTTGATTTTCTTCAATGGCTTCAAA 5 gly CEVEHWLVMDV ATCATCGTTCTGAAGTTGAACATTG (SEQ ID NO: 454) GCTTGTTATGGATGTT(SEQ ID NO: 558) mL20-1 IIQRDMSMLWEL CATCAACGTGATATGTCTATGCTTT 5 gly CLDVLDGLRQYS GGGAACTTCTTGATGTTCTTGATGG (SEQ ID NO: 439) TCTTCGTCAATATTCT(SEQ ID NO: 559) mL20-2 TQRDMSMLDGLL ACTCAACGTGATATGTCTATGCTTG 5 gly CEVLDQLRQQR ATGGTCTTCTTGAAGTTCTTGATCA (SEQ ID NO: 440) ACTTCGTCAACAACGT(SEQ ID NO: 560) mL20-3 TSRDMSLLWELL ACCTCCCGTGACATGTCCCTGCTGT 5 gly CEELDRLGHQR GGGAACTGCTGGAAGAACTGGACC (SEQ ID NO: 441) GTCTGGGTCACCAGCGT(SEQ ID NO: 561) mL20-4 MQHDMSMLYGL ATGCAACATGATATGTCTATGCTTT 5 gly CVELLESLGHQI ATGGTCTTGTTGAACTTCTTGAATC (SEQ ID NO: 442) TCTTGGTCATCAAATT(SEQ ID NO: 562) mL20-5 WNRDMRMLESL TGGAATCGTGATATGCGTATGCTTG 5 gly CFEVLDGLRQQV AATCTCTTTTTGAAGTTCTTGATGG (SEQ ID NO: 443) TCTTCGTCAACAAGTT(SEQ ID NO: 563) mL20-6 GYRDMSMLEGLL GGTTATCGTGATATGTCTATGCTTG 5 gly CAVLDRLGPQL AAGGTCTTCTTGCTGTTCTTGATCG (SEQ ID NO: 444) TCTTGGTCCACAACTT(SEQ ID NO: 564) mL20 Con1 TQRDMSMLEGLL ACTCAACGTGATATGTCTATGCTTG 5 glyC EVLDRLGQQR AAGGTCTTCTTGAAGTTCTTGATCG (SEQ ID NO: 445) TCTTGGTCAACAACGT(SEQ ID NO: 565) mL20 Con2 WYRDMSMLEGL TGGTACCGTGACATGTCCATGCTG 5 gly CLEVLDRLGQQR GAAGGTCTGCTGGAAGTTCTGGAC (SEQ ID NO: 446) CGTCTGGGTCAGCAGCGT(SEQ ID NO: 566) mL21-1 TQNSRQMLLSDF ACTCAAAATTCTCGTCAAATGCTTC 5 gly CMMLVGSMIQG TTTCTGATTTTATGATGCTTGTTGG (SEQ ID NO: 447) TTCTATGATTCAAGGT(SEQ ID NO: 567) mL21-2 MQTSRHILLSEFM ATGCAAACTTCTCGTCATATTCTTC 5 gly CMLVGSIMHG TTTCTGAATTTATGATGCTTGTTGG (SEQ ID NO: 448) TTCTATTATGCATGGT(SEQ ID NO: 568) mL21-3 HDNSRQMLLSDL CACGACAACTCCCGTCAGATGCTG 5 gly CLHLVGTMIQG CTGTCCGACCTGCTGCACCTGGTTG (SEQ ID NO: 449) GTACCATGATCCAGGGT(SEQ ID NO: 569) mL21-4 MENSRQNLLRELI ATGGAAAACTCCCGTCAGAACCTG 5 gly CMLVGNMSHQ CTGCGTGAACTGATCATGCTGGTTG (SEQ ID NO: 450) GTAACATGTCCCACCAG(SEQ ID NO: 570) mL21-5 QDTSRHMLLREF CAGGACACCTCCCGTCACATGCTG 5 gly CMMLVGEMIQG CTGCGTGAATTCATGATGCTGGTTG (SEQ ID NO: 451) GTGAAATGATCCAGGGT(SEQ ID NO: 571) mL21 Con1 DQNSRQMLLSDL GACCAGAACTCCCGTCAGATGCTG 5 gly CMILVGSMIQG CTGTCCGACCTGATGATCCTGGTTG (SEQ ID NO: 452) GTTCCATGATCCAGGGT(SEQ ID NO: 572) mTN8-19-1 VALHGQCTRWP GTTGCTCTTCATGGTCAATGTACTC 5 gly CWMCPPQREG GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 305) AACGTGAAGGT(SEQ ID NO: 573) mTN8-19-2 YPEQGLCTRWPW TATCCAGAACAAGGTCTTTGTACTC 5 glyC MCPPQTLA GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 306) AAACTCTTGCT(SEQ ID N: 574) mTN8-19-3 GLNQGHCTRWP GGTCTGAACCAGGGTCACTGCACC 5 gly CWMCPPQDSN CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 307) CAGGACTCCAAC(SEQ ID NO: 575) mTN8-19-4 MITQGQCTRWPW ATGATTACTCAAGGTCAATGTACTC 5 glyC MCPPQPSG GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 308) AACCATCTGGT(SEQ ID NO: 576) mTN8-19-5 AGAQEHCTRWP GCTGGTGCTCAGGAACACTGCACC 5 gly CWMCAPNDWI CGTTGGCCGTGGATGTGCGCTCCG (SEQ ID NO: 309) AACGACTGGATC(SEQ ID NO: 577) mTN8-19-6 GVNQGQCTRWR GGTGTTAACCAGGGTCAGTGCACC 5 gly CWMCPPNGWE CGTTGGCGTTGGATGTGCCCGCCG (SEQ ID NO: 310) AACGGTTGGGAA(SEQ ID NO: 578) mTN8-19-7 LADHGQCIRWPW CTGGCTGACCACGGTCAGTGCATC 5 gly CMCPPEGWE CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 311) GAAGGTTGGGAA(SEQ ID NO: 579) mTN8-19-8 ILEQAQCTRWPW ATCCTGGAACAGGCTCAGTGCACC 5 gly CMCPPQRGG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 312) CAGCGTGGTGGT(SEQ ID NO: 580) mTN8-19-9 TQTHAQCTRWP ACTCAAACTCATGCTCAATGTACTC 5 gly CWMCPPQWEG GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 313) AATGGGAAGGT(SEQ ID NO: 581) mTN8-19-10 VVTQGHCTLWP GTTGTTACTCAAGGTCATTGTACTC 5 glyC WMCPPQRWR TTTGGCCATGGATGTGTCCACCACA (SEQ ID NO: 314) ACGTTGGCGT(SEQ ID NO: 582) mTN8-19-11 IYPHDQCTRWPW ATTTATCCACATGATCAATGTACTC 5 glyC MCPPQPYP GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 315) AACCATATCCA(SEQ ID NO: 583) mTN8-19-12 SYWQGQCTRWP TCTTATTGGCAAGGTCAATGTACTC 5 glyC WMCPPQWRG GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 316) AATGGCGTGGT(SEQ ID NO: 584) mTN8-19-13 MWQQGHCTRWP ATGTGGCAACAAGGTCATTGTACT 5 gly CWMCPPQGWG CGTTGGCCATGGATGTGTCCACCA (SEQ ID NO: 317) CAAGGTTGGGGT(SEQ ID NO: 585) mTN8-19-14 EFTQWHCTRWP GAATTCACCCAGTGGCACTGCACC 5 gly CWMCPPQRSQ CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 318) CAGCGTTCCCAG(SEQ ID NO: 586) mTN8-19-15 LDDQWQCTRWP CTGGACGACCAGTGGCAGTGCACC 5 gly CWMCPPQGFS CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 319) CAGGGTTTCTCC(SEQ ID NO: 587) mTN8-19-16 YQTQGLCTRWP TATCAAACTCAAGGTCTTTGTACTC 5 glyC WMCPPQSQR GTTGGCCATGGATGTGTCCACCAC (SEQ ID NO: 320) AATCTCAACGT(SEQ ID NO: 588) mTN8-19-17 ESNQGQCTRWP GAATCTAATCAAGGTCAATGTACT 5 gly CWMCPPQGGW CGTTGGCCATGGATGTGTCCACCA (SEQ ID NO: 321) CAAGGTGGTTGG(SEQ ID NO: 589) mTN8-19-18 WTDRGPCTRWP TGGACCGACCGTGGTCCGTGCACC 5 gly CWMCPPQANG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 322) CAGGCTAACGGT(SEQ ID NO: 590) mTN8-19-19 VGTQGQCTRWP GTTGGTACCCAGGGTCAGTGCACC 5 gly CWMCPPYETG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 323) TACGAAACCGGT(SEQ ID NO: 591) mTN8-19-20 PYEQGKCTRWP CCGTACGAACAGGGTAAATGCACC 5 gly CWMCPPYEVE CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 324) TACGAAGTTGAA(SEQ ID NO: 592) mTN8-19-21 SEYQGLCTRWPW TCCGAATACCAGGGTCTGTGCACC 5 glyC MCPPQGWK CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 325) CAGGGTTGGAAA(SEQ ID NO: 593) mTN8-19-22 TFSQGHCTRWPW ACCTTCTCCCAGGGTCACTGCACCC 5 glyC MCPPQGWG GTTGGCCGTGGATGTGCCCGCCGC (SEQ ID NO: 326) AGGGTTGGGGT(SEQ ID NO: 594) mTN8-19-23 PGAHDHCTRWP CCGGGTGCTCACGACCACTGCACC 5 gly CWMCPPQSRY CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 327) CAGTCCCGTTAC(SEQ ID NO: 595) mTN8-19-24 VAEEWHCRRWP GTTGCTGAAGAATGGCACTGCCGT 5 gly CWMCPPQDWR CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 328) CAGGACTGGCGT(SEQ ID NO: 596) mTN8-19-25 VGTQGIICTRWP GTTGGTACCCAGGGTCACTGCACC 5 glyC WMCPPQPAG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 329) CAGCCGGCTGGT(SEQ ID NO: 597) mTN8-19-26 EEDQAHCRSWP GAAGAAGACCAGGCTCACTGCCGT 5 gly CWMCPPQGWV TCCTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 330) CAGGGTTGGGTT(SEQ ID NO: 598) mTN8-19-27 ADTQGHCTRWP GCTGACACCCAGGGTCACTGCACC 5 gly CWMCPPQHWF CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 331) CAGCACTGGTTC(SEQ ID NO: 599) mTN8-19-28 SGPQGHCTRWPW TCCGGTCCGCAGGGTCACTGCACC 5 glyC MCAPQGWF CGTTGGCCGTGGATGTGCGCTCCG (SEQ ID NO: 332) CAGGGTTGGTTC(SEQ ID NO: 600) mTN8-19-29 TLVQGHCTRWP ACCCTGGTTCAGGGTCACTGCACC 5 gly CWMCPPQRWV CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 333) CAGCGTTGGGTT(SEQ ID NO: 601) mTN8-19-30 GMAHGKCTRWA GGTATGGCTCACGGTAAATGCACC 5 gly CWMCPPQSWK CGTTGGGCTTGGATGTGCCCGCCG (SEQ ID NO: 334) CAGTCCTGGAAA(SEQ ID NO: 602) mTN8-19-31 ELYHGQCTRWP GAACTGTACCACGGTCAGTGCACC 5 gly CWMCPPQSWA CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 335) CAGTCCTGGGCT(SEQ ID NO: 603) mTN8-19-32 VADHGHCTRWP GTTGCTGACCACGGTCACTGCACC 5 gly CWMCPPQGWG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 336) CAGGGTTGGGGT(SEQ ID NO: 604 mTN8-19-33 PESQGHCTRWPW CCGGAATCCCAGGGTCACTGCACC 5 gly CMCPPQGWG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 337) CAGGGTTGGGGT(SEQ ID NO: 605) mTN8-19-34 IPAHGHCTRWPW ATCCCGGCTCACGGTCACTGCACC 5 glyC MCPPQRWR CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 338) CAGCGTTGGCGT(SEQ ID NO: 606) mTN8-19-35 FTVHGHCTRWP TTCACCGTTCACGGTCACTGCACCC 5 glyC WMCPPYGWV GTTGGCCGTGGATGTGCCCGCCGT (SEQ ID NO: 339) ACGGTTGGGTT(SEQ ID NO: 607) mTN8-19-36 PDFPGHCTRWRW CCAGATTTTCCAGGTCATTGTACTC 5 glyC MCPPQGWE GTTGGCGTTGGATGTGTCCACCAC (SEQ ID NO: 340) AAGGTTGGGAA(SEQ ID NO: 608) mTN8-19-37 QLWQGPCTQWP CAGCTGTGGCAGGGTCCGTGCACC 5 gly CWMCPPKGRY CAGTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 341) AAAGGTCGTTAC(SEQ ID NO: 609) mTN8-19-38 HANDGHCTRWQ CACGCTAACGACGGTCACTGCACC 5 gly CWMCPPQWGG CGTTGGCAGTGGATGTGCCCGCCG (SEQ ID NO: 342) CAGTGGGGTGGT(SEQ ID NO: 610) mTN8-19-39 ETDHGLCTRWPW GAAACCGACCACGGTCTGTGCACC 5 glyC MCPPYGAR CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 343) TACGGTGCTCGT(SEQ ID NO: 611) mTN8-19-40 GTWQGLCTRWP GGTACCTGGCAGGGTCTGTGCACC 5 gly CWMCPPQGWQ CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 344) CAGGGTTGGCAG(SEQ ID NO: 612) mTN8-19 Con1 VATQGQCTRWP GTTGCTACCCAGGGTCAGTGCACC 5 glyC WMCPPQGWG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 345) CAGGGTTGGGGT(SEQ ID NO: 613) mTN8 19 Con2 VATQGQCTRWP GTTGCTACCCAGGGTCAGTGCACC 5 glyC WMCPPQRWG CGTTGGCCGTGGATGTGCCCGCCG (SEQ ID NO: 346) CAGCGTTGGGGT(SEQ ID NO: 614) 2X mTN8-19-7 FC-5G-AQ- CTTGCTGATCATGGTCAATGTATTC 1K CLADHGQCIRWPW GTTGGCCATGGATGTGTCCACCAG MCPPEGWELEGSAAGGTTGGGAACTCGAGGGTTCCG GSATGGSGSTASS GTTCCGCTACCGGCGGCTCTGGCTCGSGSATGLADHG CACTGCTTCTTCCGGTTCCGGTTCT QCIRWPWMCPPEGCTACTGGTCTGGCTGACCACGGT GWE-LE CAGTGCATCCGTTGGCCGTGGATG(SEQ ID NO: 615) TGCCCGCCGGAAGGTTGGGAACTG GAA (SEQ ID NO: 616)2X mTN8-19-7 FC-5G-AQ- CTTGCTGATCATGGTCAATGTATTC 1K C ST GG del2xLADHGQCIRWPW GTTGGCCATGGATGTGTCCACCAG LE MCPPEGWEGSGSAAGGTTGGGAAGGTTCCGGTTCCG ATGGSGGGASSG CTACCGGCGGCTCTGGCGGTGGCGSGSATGLADHGQ CTTCTTCCGGTTCCGGTTCTGCTAC CIRWPWMCPPEGTGGTCTGGCTGACCACGGTCAGTG WE CATCCGTTGGCCGTGGATGTGTCCA (SEQ ID NO: 617)CCAGAAGGTTGGGAA (SEQ ID NO: 618) 2X mTN8-19-21 FC-5G-AQ-TCTGAATATCAAGGTCTTTGTACTC 1K C SEYQGLCTRWPW GTTGGCCATGGATGTGTCCACCACMCPPQGWKLEGS AAGGTTGGAAACTCGAGGGTTCCG GSATGGSGSTASSGTTCCGCTACCGGCGGCTCTGGCTC GSGSATGSEYQG CACTGCTTCTTCCGGTTCCGGTTCTLCTRWPWMCPPQ GCTACTGGTTCTGAGTATCAAGGC GWK-LE CTCTGTACTCGCTGGCCATGGATGT(SEQ ID NO: 619) GTCCACCACAAGGCTGGAAGCTGG AA (SEQ ID NO: 620)2X mTN8-19-21 FC-5G-AQ- TCTGAATATCAAGGTCTTTGTACTC 1K C ST-GG del2xSEYQGLCTRWPW GTTGGCCATGGATGTGTCCACCAC LE MCPPQGWKGSGSAAGGTTGGAAAGGTTCCGGTTCCG ATGGSGGGASSG CTACCGGCGGCTCTGGCGGTGGCGSGSATGSEYQGL CTTCTTCCGGTTCCGGTTCTGCTAC CTRWPWMCPPQTGGTTCTGAGTATCAAGGCCTCTGT GWK ACTCGCTGGCCATGGATGTGTCCA (SEQ ID NO: 621)CCACAAGGTTGGAAA (SEQ ID NO: 622) 2X mTN8-19-22 FC-5G-AQ-ACTTTTTCTCAAGGTCATTGTACTC 1K C TFSQGHCTRWPW GTTGGCCATGGATGTGTCCACCACMCPPQGWGLEGS AAGGTTGGGGTCTCGAGGGTTCCG GSATGGSGSTASSGTTCCGCTACCGGCGGCTCTGGCTC GSGSATGTFSQG CACTGCTTCTTCCGGTTCCGGTTCTHCTRWPWMCPP GCTACTGGTACTTTTTCTCAAGGCC QGWG-L E ATTGTACTCGCTGGCCATGGATGTG(SEQ ID NO: 623) TCCACCACAAGGCTGGGGCCTGGA A (SEQ ID NO: 624)2X mTN8-19-32 FC-5G-AQ- GTTGCTGATCATGGTCATTGTACTC 1K C VADHGHCTRWPGTTGGCCATGGATGTGTCCACCAC WMCPPQGWGLE AAGGTTGGGGTCTCGAGGGTTCCGGSGSATGGSGST GTTCCGCAACCGGCGGCTCTGGCT ASSGSGSATGVACCACTGCTTCTTCCGGTTCCGGTTC DHGHCTRWPWM TGCTACTGGTGTTGCTGACCACGGTCPPQGWG-LE CACTGCACCCGTTGGCCGTGGATG (SEQ ID NO: 625)TGCCCGCCGCAGGGTTGGGGTCTG GAA (SEQ ID NO: 626) 2X mTN8-19-32 FC-5G-AQ-GTTGCTGATCATGGTCATTGTACTC 1K C ST-GG del2x VADHGHCTRWPGTTGGCCATGGATGTGTCCACCAC LE WMCPPQGWGGS AAGGTTGGGGTGGTTCCGGTTCCGGSATGGSGGGAS CTACCGGCGGCTCTGGCGGTGGTG SGSGSATGVADIICTTCTTCCGGTTCCGGTTCTGCTAC GHCTRWPWVCPP TGGTGTTGCTGACCACGGTCACTGC QGWGACCCGTTGGCCGTGGGTGTGTCCA (SEQ ID NO: 627) CCACAAGGTTGGGGT(SEQ ID NO: 628) 2X mTN8-19-33 FC-5G-AQ- CCAGAATCTCAAGGTCATTGTACTC 1K CPESQGHCTRWPW GTTGGCCATGGATGTGTCCACCAC MCPPQGWGLEGSAAGGTTGGGGTCTCGAGGGTTCCG GSATGGSGSTASS GTTCCGCTACCGGCGGCTCTGGCTCGSGSATGPESQG CACTGCTTCTTCCGGTTCCGGTTCT HCTRWPWMCPPGCTACTGGTCCGGAATCCCAGGGT QGWGLE CACTGCACCCGTTGGCCGTGGATG(SEQ ID NO: 629) TGCCCGCCGCAGGGTTGGGGTCTG GAA (SEQ ID NO: 630)2X mTN8 19 33 FC 5G AQ CCAGAATCTCAAGGTCATTGTACTC 1K C ST-GG del2xPESQGHCTRWPW GTTGGCCATGGATGTGTCCACCAC LE MCPPQGWGGSGSAAGGTTGGGGTGGTTCCGGTTCCG ATGGSGGGASSG CTACCGGCGGCTCTGGCGGTGGTGSGSATGPESQGH CTTCTTCCGGTTCCGGTTCTGCTAC CTRWPWMCPTGGTCCGGAATCCCAGGGTCACTG PQGWG CACCCGTTGGCCGTGGATGTGTCC (SEQ ID NO: 631)ACCACAAGGTTGGGGT (SEQ ID NO: 632)

Example 7 In Vitro Screening of Affinity Matured Peptibodies

The following exemplary peptibodies were screened according to theprotocols set forth above to obtain the following K_(D) and IC₅₀ values.Table VII shows the range of K_(D) values for selected affinity maturedpeptibodies compared with the parent peptibodies, as determined byKinExA™ solution based assays or BIAcore® assays. These valuesdemonstrate increased binding affinity of the affinity maturedpeptibodies for myostatin compared with the parent peptibodies. TableVIII shows IC₅₀ values for a number of affinity matured peptibodies. Arange of values is given in this table.

TABLE VII peptibodies K_(D) TN8-19 (parent) >1 nM 2xmTN8-19 (parent) >1nM 1x mTN8-19-7 10 pM 2x mTN8-19-7 12 pM 1x mTN8-19-21  6 pM 2xmTN8-19-21  6 pM 1x mTN8-19-32  9 pM 1x mTN8-19-33 21 pM 2x mTN8-19-33 3 pM 1x mTN8-19-22  4 pM 1x mTN8-19-con1 20 pM

TABLE VIII Affinity Matured Peptibody IC₅₀ (nM) mTN8-19 Con1 1.0-4.4mTN8-19-2 7.508-34.39 mTN8-19-4 16.74 mTN8-19-5 7.743 3.495 mTN8-19-617.26 mTN8-19-7 1.778 mTN8-19-9 22.96-18.77 mTN8-19-10 5.252-7.4 mTN8-19-11 28.66 mTN8-19-12 980.4 mTN8-19-13 20.04 mTN8-19-14 4.0656.556 mTN8-19-16 4.654 mTN8-19-21 2.767-3.602 mTN8-19-22 1.927-3.258mTN8-19-23 6.584 mTN8-19-24 1.673-2.927 mTN8-19-27 4.837-4.925mTN8-19-28 4.387 mTN8-19-29 6.358 mTN8-19-32 1.842-3.348 mTN8-19-332.146-2.745 mTN8-19-34 5.028-5.069 mTN8Con6-3 86.81 mTN8Con6-5 2385mTN8-19-7(-LE)  1.75-2.677 mTN8-19-21(-LE) 2.49 mTN8-19-33(-LE) 1.8082xmTN8-19-7 0.8572-2.649  2xmTN8-19-9 1.316-1.228 2xmTN8-19-14 1.18-1.322 2xmTN8-19-16 0.9903-1.451  2xmTN8-19-21 0.828-1.4342xmTN8-19-22 0.9937-1.22  2xmTN8-19-27 1.601-3.931 2xmTN8-19-7(-LE)1.077-1.219 2xmTN8-19-21(-LE) 0.8827-1.254  2xmTN8-19-33(-LE) 1.12-1.033 mL2-7 90.24 mL2-9 105.5 mL15-7 32.75 mL15-9 354.2 mL20-2122.6 mL20-3 157.9 mL20-4 160

Example 8 In Vivo Anabolic Activity of Exemplary Peptibodies

The CD1 nu/nu mouse model (Charles River Laboratories, Massachusettes)was used to determine the in vivo efficacy of the peptibodies of thepresent invention which included the human Fc region (huFc). This modelresponded to the inhibitors of the present invention with a rapidanabolic response which was associated with increased dry muscle massand an increase in myofibrillar proteins but was not associated withaccumulation in body water content.

In one example, the efficacy of 1× peptibody mTN8-19-21 in vivo wasdemonstrated by the following experiment. A group of 10 8 week old CD1nu/nu mice were treated twice weekly or once weekly with dosages of 1mg/kg, 3 mg/kg and 10 mg/kg (subcutaneous injection). The control groupof 10 8 week old CD1 nu/nu mice received a twice weekly (subcutaneous)injection of huFc (vehicle) at 10 mg/kg. The animals were weighed everyother day and lean body mass determined by NMR on day 0 and day 13. Theanimals are then sacrificed at day 14 and the size of the gastrocnemiusmuscle determined. The results are shown in FIGS. 2 and 3. FIG. 2 showsthe increase in total body weight of the mice over 14 days for thevarious dosages of peptibody compared with the control. As can be seenfrom FIG. 2, all of the dosages have show an increase in body weightcompared with the control, with all of the dosages showing statisticallysignificant increases over the control by day 14. FIG. 3 shows thechange in lean body mass on day 0 and day 13 as determined by NMR, aswell as the change in weight of the gastrocnemius muscle dissected fromthe animals at day 14.

In another example, the 1× mTN8-19-32 peptibody was administered to CD1nu/nu mice in a biweekly injection of 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30mg/kg compared with the huFc control (vehicle). The peptibody-treatedanimals show an increase in total body weight (not shown) as well aslean body mass on day 13 compared with day 0 as determined by NMRmeasurement. The increase in lean body mass is shown in FIG. 4.

In another example, a 1× affinity-matured peptibody was compared with a2× affinity-matured peptibody for in vivo anabolic efficacy. CD1 nu/numale mice (10 animals per group) were treated with twice weeklyinjections of 1 mg/kg and 3 mg/kg of 1× mTN8-19-7 and 2× mTN8-19-7 for35 days, while the control group (10 animals) received twice weeklyinjections of huFc (3 mg/kg). As shown in FIG. 5, treatment with the 2×peptibody resulted in a greater body weight gain and lean carcass weightat necropsy compared with the 1× peptibody or control.

Example 9 Increase in Muscular Strength

Normal age-matched 4 month old male C57B/6 mice were treated for 30 dayswith 2 injections per week subcutaneous injections 5 mg/kg per week of2× mTN8-19-33, 2× mTN8-19-7, and huFc vehicle control group (10animals/group). The animals were allowed to recover without any furtherinjections. Gripping strength was measured on day 18 of the recoveryperiod. Gripping strength was measured using a Columbia Instrumentsmeter, model 1027 dsm (Columbus, Ohio). Peptibody treatment resulted insignificant increase in gripping strength, with 2× mTN8-19-33 pretreatedanimals showing a 14% increase in gripping strength compared with thecontrol-treated mice, while 2× mTN8-19-7 showed a 15% increase ingripping strength compared with the control treated mice.

Example 10 Pharmacokinetics

In vivo pharmacokinetics experiments were performed using representativepeptibodies without the LE sequences. 10 mg/kg and 5 mg/kg dosages wereadministered to CD1 nu/nu mice and the following parameters determined.Cmax (ug/mL), area under the curve (AUC) (ug-hr/mL), and half-life (hr).It was found that the 2× versions of the affinity matured peptibodieshave a significantly longer half-life than the 1× versions. For example,1× affinity matured mTN8-19-22 has a half-life in the animals of about50.2 hours, whereas 2× mTN8-19-22 has a half-life of about 85.2 hours.Affinity matured 1× mTN8-7 has a half-life of about 65 hours, whereas 2×mTN8-19-7 has a half-life of about 106 hours.

Example 11 Treatment of Mdx Mice

The peptibodies of the present invention have been shown to increaselean muscle mass in an animal and are useful for the treatment of avariety of disorders which involve muscle wasting. Muscular dystrophy isone of those disorders. The mouse model for Duchenne's musculardystrophy is the Duchenne mdx mouse (Jackson Laboratories, Bar Harbor,Me.). Aged (10 month old) mdx mice were injected with either thepeptibody 1× mTN8-19-33 (n=8/group) or with the vehicle huFc protein(N=6/group) for a three month period of time. The dosing schedule wasevery other day, 10 mg/kg, by subcutaneous injection. The peptibodytreatment had a positive effect on increasing and maintaining body massfor the aged mdx mice. Significant increases in body weight wereobserved in the peptibody-treated group compared to the hu-Fc-treatedcontrol group, as shown in FIG. 6A. In addition, NMR analysis revealedthat the lean body mass to fat mass ratio was also significantlyincreased in the aged mdx mice as a result of the peptibody treatmentcompared with the control group, and that the fat percentage of bodyweight decreased in the peptibody treated mice compared with the controlgroup, as shown in FIG. 6B.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components of the invention. Indeed, various modificationsof the invention, in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

What is claimed is:
 1. A binding agent capable of binding myostatincomprising the structure F¹-(L¹)-P¹ or multimers thereof, wherein F¹ isa human IgG Fc, wherein L¹ is (Gly)₅, wherein the binding agent furthercomprises AQ and wherein P¹ is SEQ ID NO:
 311. 2. A pharmaceuticalcomposition comprising a therapeutically effective amount of the bindingagent of claim 1 in admixture with a pharmaceutically acceptable carrierthereof.
 3. An isolated nucleic acid molecule comprising apolynucleotide encoding a monomer of the binding agent of claim
 1. 4.The nucleic acid molecule of claim 3, wherein the polynucleotide is SEQID NO:579.
 5. An expression vector comprising the polynucleotide ofclaim
 3. 6. A host cell comprising the expression vector of claim
 5. 7.A method of making a myostatin binding agent comprising culturing thehost cell of claim 6 under conditions suitable for expressing thebinding agent, and purifying the binding agent.
 8. A method ofinhibiting myostatin activity in a subject comprising administering aneffective amount of the composition of claim 2 to the subject.
 9. Amethod of increasing lean muscle mass in a subject comprisingadministering the composition of claim 2 to the subject.
 10. A method ofincreasing the ratio of lean muscle mass to fat in a subject comprisingadministering a therapeutically effective amount of the composition ofclaim 2 to the subject.
 11. A method of treating a muscle-wastingdisease in a subject comprising administering a therapeuticallyeffective amount of the composition of claim 2 to the subject.
 12. Themethod of claim 11, wherein the disease is selected from musculardystrophy, amyotrophic lateral sclerosis, congestive obstructivepulmonary disease, chronic heart failure, cancer, AIDs, renal failure,uremia, rheumatoid arthritis, age-related sarcopenia, and muscle-wastingdue to prolonged bedrest, spinal chord injury, stroke, bone fracture,and aging.
 13. A method of detecting or measuring myostatin in a samplecomprising contacting the sample with the binding agent of claim 1 anddetecting or measuring the bound complex.
 14. The binding agent of claim1, wherein F¹ comprises the sequence of SEQ ID NO:296.
 15. The bindingagent of claim 1, wherein (L¹)-P¹ comprises the sequence SEQ ID NO:617.16. A binding agent capable of binding myostatin comprising a dimer of apolypeptide with the structure F¹-(L¹)-P¹, wherein F¹ a human IgG Fccomprising the sequence of SEQ ID NO:296 and L¹ is (Gly)₅, wherein thebinding agent further comprises AQ and P¹ is SEQ ID NO:
 311. 17. Apharmaceutical composition comprising a therapeutically effective amountof the binding agent of claim 16 in admixture with a pharmaceuticallyacceptable carrier thereof.
 18. An isolated nucleic acid moleculecomprising a polynucleotide encoding a monomer of the binding agent ofclaim
 16. 19. An expression vector comprising the polynucleotide ofclaim
 18. 20. A host cell comprising the expression vector of claim 19.21. A host cell comprising the expression vector of claim 19, whereinthe host cell is an E. coli cell.
 22. A method of making a myostatinbinding agent comprising culturing the host cell of claim 21 underconditions suitable for expressing the binding agent, and purifying thebinding agent.
 23. A method of inhibiting myostatin activity in asubject comprising administering an effective amount of the compositionof claim 17 to the subject.
 24. A method of increasing lean muscle massin a subject comprising administering the composition of claim 17 to thesubject.
 25. A method of increasing the ratio of lean muscle mass to fatin a subject comprising administering a therapeutically effective amountof the composition of claim 17 to the subject.
 26. A method of treatinga muscle-wasting disease in a subject comprising administering atherapeutically effective amount of the composition of claim 17 to thesubject.
 27. The method of claim 26, wherein the disease is selectedfrom muscular dystrophy, amyotrophic lateral sclerosis, congestiveobstructive pulmonary disease, chronic heart failure, cancer, AIDs,renal failure, uremia, rheumatoid arthritis, age-related sarcopenia, andmuscle-wasting due to prolonged bedrest, spinal chord injury, stroke,bone fracture, and aging.
 28. A method of detecting or measuringmyostatin in a sample comprising contacting the sample with a bindingagent of claim 16 and detecting or measuring the bound complex.